Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 653 - 5 Crystallization and preliminary X-ray analysis of nitrous oxide reductase from Paracoccus pantotrophus; Jafferji A et al.; Nitrous oxide reductase is a periplasmic respiratory protein with a novel copper catalytic centre; it catalyses the terminal step, reduction of nitrous oxide to nitrogen, of the bacterial denitrification process . Nitrous oxide reductase from Paracoccus pantotrophus has been crystallized by the hanging-drop method . A prerequisite for crystallization was the oxidation of the enzyme with potassium ferricyanide in order to obtain homogenous oxidation states of the copper centres . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 116.4, b = 118.3, c = 267.0 A . Two homodimers, of approximate molecular weight 67 kDa per subunit, probably constitute the asymmetric unit and give a Matthews coefficient, V(m), of 3.4 A(3) Da(-1) and a solvent content of 59% by volume . The crystals diffract X-rays to 3.0 A resolution on an in-house source and are suitable for structure determination. Biochemistry, 2000 Apr 25, 39(16), 4929 - 38 Unusual spectroscopic and electrochemical properties of the 2{4Fe-4S} ferredoxin of Thauera aromatica; Boll M et al.; A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica . It contains two {4Fe-4S} clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension . The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both {4Fe-4S} clusters from T . aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR . Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE . X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced {4Fe-4S} cluster . No typical S = (1)/(2) EPR signals were observed . At lower potentials (less than -500 mV), the more negative {4Fe-4S} cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin {4Fe-4S} cluster with a g(av) of 1.94 . However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals . At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2) . The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the {4Fe-4S} clusters . The observation of different spin states in T . aromatica ferredoxin is novel among CvFd-type ferredoxins. J Biol Chem, 2000 Jul 21, 275(29), 21889 - 95 Nonaromatic products from anoxic conversion of benzoyl-CoA with benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase; Boll M et al.; The enzymes benzoyl-CoA reductase and cyclohex-1, 5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica . Reaction products obtained with {ring-(13)C(6)}benzoyl-CoA and {ring-(14)C}benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy . The main product obtained with titanium(III) citrate or with reduced {8Fe-8S}-ferredoxin from T . aromatica as electron donors was identified as cyclohexa-1, 5-diene-1-carbonyl-CoA . The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase . Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA . The potential required for the first electron transfer to the model compound S-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as -1.9 V versus a standard hydrogen electrode . The energetics of enzymatic ring reduction of benzoyl-CoA are discussed. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 909 - 15 Differentiation of newly described antarctic bacterial isolates related to Roseobacter species based on 16S-23S rDNA internal transcribed spacer sequences; Soller R et al.; The 16S-23S rDNA internal transcribed spacer (ITS) of Roseobacter denitrificans, Roseobacter litoralis, Ruegeria algicola and strains of the recently described species Antarctobacter heliothermus and Roseovarius tolerans were analysed in order to examine DNA sequence variations and to draw conclusions about inter- and intraspecific relationships . A . heliothermus included four strains with an ITS fragment length of 1092 bp . Roseovarius tolerans was described on the basis of eight strains . Five of these harboured two ITS fragments of different lengths (959 and about 1100 bp), while the others had one fragment of either 1083 bp (two strains) or 1165 bp (one strain) . ITS lengths of the related species Roseobacter denitrificans, Roseobacter litoralis and Ruegeria algicola were found to be 980, 984 and 1158 bp, respectively . Phylogenetic analyses of the DNA sequences allowed species affiliation of strains with sequence length differences of > 200 bp and recognition of relationships based on a well-supported ITS tree . The strains of A . heliothermus and Roseovarius tolerans each formed a monophyletic branch and they were separated from each other by Ruegeria algicola . This species was now clearly separated from Roseobacter denitrificans and Roseobacter litoralis, which corresponded to the new genus affiliation of Ruegeria algicola . These data were additionally supported by analyses of the structure, relative position and order of genes for tRNA(Ile) and tRNA(Ala) found within the ITS of each strain . Comparative DNA sequence analyses of ITS and 16S rDNA revealed limitations, on species and strain levels, with respect to the phylogenetic resolution of the 16S rDNA due to the limited number of informative (variable) sites, while ITS sequence analyses provided more variable and sufficiently conserved positions to discriminate between strains and to reconstruct their taxonomic relationships. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 547 - 50 Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain; Kelly DP et al.; Thiobacillus denitrificans is physiologically similar to the type species of the genus Thiobacillus, Thiobacillus Thioparus, and both are located in the beta-subclass of the Proteobacteria . T . denitrificans is distinguished from all other Thiobacillus species by its ability to grow as a facultatively anaerobic chemolithotroph, coupling the oxidation of inorganic sulfur compounds to the reduction of nitrate, nitrite and other oxidized nitrogen compounds to dinitrogen . A definitive description of this species is provided and strain NCIMB 9548T is designated as the type strain of the species, thereby correcting an earlier error in the literature. Acta Microbiol Pol, 1999, 48(3), 297 - 306 Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures; Son TT et al.; Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa . In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions . As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively . Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively . Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L. Appl Environ Microbiol, 2000 Apr, 66(4), 1595 - 601 Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions; Rockne KJ et al.; Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene . The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate . Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4 . Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay . Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite . Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent . No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls . Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added (14)C-labeled naphthalene . After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites . Nitrate consumption, along with the results from the (14)C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4 . Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius . This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures. Appl Environ Microbiol, 2000 Apr, 66(4), 1564 - 71 Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples; Michotey V et al.; Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples . The specificity and sensitivity of these primers were tested . Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques . Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method . Enumerations using both molecular techniques yielded similar results in seawater and sediment samples . However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting. Appl Environ Microbiol, 2000 Apr, 66(4), 1360 - 8 Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis; Fernandez L et al.; The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme . The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues . The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified . The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases . The protein was overproduced up to 170-fold in L . lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria . The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector . This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene . This confirms that EstA is the main enzyme responsible for esterase activity in L . lactis . Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids . Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently . Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes . We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway. Appl Environ Microbiol, 2000 Apr, 66(4), 1286 - 91 Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol; Shinoda Y et al.; Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp . strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively . Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process . Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions . Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group . Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis . It grew on phenol only under denitrifying conditions . Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features. J Bacteriol, 2000 Apr, 182(8), 2179 - 83 Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8; Ramirez S et al.; Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration . In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell . We describe the presence of two genes, narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters . The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters . To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes . Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability . Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type . These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters . The possible differential roles of these proteins in vivo are discussed. FEBS Lett, 2000 Mar 24, 470(2), 216 - 20 Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui; Yoshimatsu K et al.; Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state . The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide . The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex . Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate . The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl. Biochemistry, 2000 Mar 21, 39(11), 2989 - 96 Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a; Lee HM et al.; As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient . X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels . The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase {Yoshikawa et al . (1998) Science 280, 1723-1729} . In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis . Mutants were generated in eight different sites and the enzymes have been purified and characterized . The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans {Pfitzner et al . (1998) J . Biomembr . Bioenerg . 30, 89-93} . Each of the mutants in R . sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes . This includes H456A, where in the P . denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme . The only mutations that result in completely inactive enzyme in the set examined in the R . sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures . To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained . The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes . Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data . In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity . The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group . Unlike R52, however, Q471 does not appear to be critical for the enzyme function. Microbiology, 2000 Feb, 146 ( Pt 2), 509 - 16 Transcriptional analysis of the nirS gene, encoding cytochrome cd1 nitrite reductase, of Paracoccus pantotrophus LMD 92.63; Saunders NF et al.; The gene for cytochrome cd1 nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS . This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d1 cofactor (nirE) . Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic . The lack of a transcript under aerobic conditions argues against a role for cytochrome cd1 in the previously proposed aerobic denitrification pathway in Pa . pantotrophus . A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified . The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant . The transcript start point for nirS has been determined by two complementary techniques, 5'-RACE (Rapid amplification of cDNA 5' ends) and primer extension . It is 29 bp upstream of the AUG of nirS . An anaerobox, which presumably binds Nnr, is centred a further 41.5 bp upstream of the transcript start . No standard sigma70 DNA sequence motifs can be identified, but a conserved sequence (T-T-GIC-C-G/C-G/C) can be found in approximately the same position (-16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide. Anal Biochem, 2000 Mar 15, 279(2), 202 - 8 Analysis of nitrite/nitrate in biological fluids: denitrification of 2-nitropropane in F344 rats; Sohn OS et al.; 2-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats . To obtain evidence that denitrification of 2-NP also occurs in vivo, we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction . However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na(2)CO(3), and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H(3)PO(4) . With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment . Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 microM (PB-pretreated) and 940 microM (vehicle-pretreated controls) . The PB-pretreated and control rats, respectively, excreted 250 and 120 micromol nitrate/nitrite in the 24-h urine post 2-NP treatment . The modifications described make the method more specific, reproducible, and more widely applicable . Biosci Biotechnol Biochem, 2000 Jan, 64(1), 61 - 71 Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster; Hayashi NR et al.; Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila) . cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria . cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins . When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE . However, the cell extract had slightly higher activity than a cell extract of E . coli without cbbA . Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H . thermoluteolus was in class IIA . Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions . The activities of the two enzymes were regulated independently. Appl Environ Microbiol, 2000 Mar, 66(3), 1209 - 12 Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans; Barak Y et al.; Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions . Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source . Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source . By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P . denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches . Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers. Appl Environ Microbiol, 2000 Mar, 66(3), 1167 - 74 Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis; Layton AC et al.; The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis . 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates . Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones . Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources . Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species . This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp . strain M3 isolated from this treatment plant . A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp . strain M3 . Phylogenetic analysis revealed that Hyphomicrobium sp . strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II . Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp . strain M3 . The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity) . Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure . Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold . Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge. Appl Environ Microbiol, 2000 Mar, 66(3), 1147 - 51 Transcriptional analysis of the tutE tutFDGH gene cluster from Thauera aromatica strain T1; Coschigano PW; The denitrifying strain T1, identified as Thauera aromatica, is able to grow with toluene serving as its sole carbon source . Previous work identified two genes, tutD and tutE, that are involved in toluene metabolism . Two small open reading frames, tutF and tutG, which may also play a role in toluene metabolism, were also identified . The present work examines the transcriptional organization and regulation of these toluene utilization genes . Northern analysis indicates that the four genes are organized into two operons, tutE and tutFDG, and that both operons are regulated in response to toluene . Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the tutE translational start and 76 bp upstream of the tutF translational start . Furthermore, a fifth gene, tutH, has been identified immediately downstream of tutG . It is transcribed from the same start site as tutFDG and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da . The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins. Biochemistry, 2000 Feb 15, 39(6), 1356 - 63 Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans . Evidence from FTIR difference spectroscopy and site-directed mutagenesis; Behr J et al.; By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates {Behr, J., Hellwig, P., Mantele, W., and Michel, H . (1998) Biochemistry 37, 7400-7406} . To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I . The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy . Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared . Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase . The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor. Eur J Biochem, 2000 Feb, 267(4), 993 - 1000 Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase; Pacheco-Moises F et al.; Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied . Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin . Among the oxyanions, sulfite induced the higher increase in ATPase activity . Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax . There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme . Dithiothreitol did not affect the ATPase activity . ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin . In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin . It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin. Chemosphere, 2000 Mar, 40(5), 549 - 55 Biological denitrification in a closed seawater system; Grguric G et al.; Build-up of high nitrate concentrations in closed seawater systems where primary productivity is undesirable and water changes are impractical presents unique problems . Nitrate concentration in Ocean Tank at the New Jersey State Aquarium reached 9500 microM after 6 years of operation . A biological denitrification system was installed in 1998 and nitrate concentration in the aquarium decreased to 7000 microM within the first 100 days of operation . The system offers additional benefits by increasing the pH and alkalinity of seawater and providing a reducing environment to balance the oxidizing disinfection environment in the aquarium . The initial performance of the denitrification system was monitored and two semi-empirical models were developed: one based on the actual methanol additions, and another based on the daily amounts of nitrogen gas removed . The first model predicts a net nitrate decrease of 39 microM/day in the aquarium . The second model predicts a net decrease of 25 microM/day, in good agreement with the empirical value of 23 microM/day . This indicates that nitrogen gas removal is the controlling factor during denitrification in this facility, and the second model can be used to predict and optimize the operation of the system. Appl Environ Microbiol, 2000 Feb, 66(2), 493 - 8 Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions; Chayabutra C et al.; A strategy for sequential hydrocarbon bioremediation is proposed . The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand . The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate . To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation . The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first . Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting . The effects of different nitrate and nitrite concentrations were examined next . When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations . However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth . Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter) . Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions . Sequential hexadecane biodegradation by P . aeruginosa was then investigated . The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations . The culture was then supplemented with nitrate and purged with nitrogen (N(2)) . Nitrate was consumed rapidly initially . The live cell concentration, however, also decreased . The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period . Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 23 - 9 Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression; Revers LF et al.; Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype . Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected . ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins. Arch Microbiol, 2000 Jan, 173(1), 58 - 64 Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria; Ehrenreich P et al.; The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor . Three distinct types of denitrifying bacteria were isolated in pure culture . A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C6H14) . Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C8H18) . A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C16H34) . Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains . Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen. J Biol Chem, 2000 Jan 21, 275(3), 1691 - 8 "ADP sulfurylase" from Thiobacillus denitrificans is an adenylylsulfate:phosphate adenylyltransferase and belongs to a new family of nucleotidyltransferases; Bruser T et al.; During AMP-dependent sulfite oxidation by some sulfur bacteria, the liberation of sulfate from adenosine-5'-phosphosulfate (APS) is catalyzed by APS:phosphate adenylyltransferase (APAT) . Here we report the first biochemical and genetic characterization of APAT . We isolated this enzyme from the chemolithoautotroph Thiobacillus denitrificans and cloned the corresponding gene . The enzyme is homodimeric with 41,387-Da subunits and exhibits a specific activity of 2100 micromol min(-1) mg(-1) . The K(m) values are K(m(APS)) = 300 microM and K(m(P(i))) = 12 mM . Catalysis occurs by a ping-pong mechanism with a covalently bound AMP as reaction intermediate . The arsenolysis of APS, but not of ADP, CDP, GDP, UDP, or IDP, is also catalyzed, indicating a specific and unidirectional function . The former enzyme name ADP-sulfurylase implies that the reverse reaction is catalyzed; therefore, this name should not be used any longer . Histidine modification of APAT results in complete inactivation that can be suppressed by substrate addition . APAT is highly similar to galactose-1-phosphate uridylyltransferase and also related to Ap(4)A phosphorylase . Active site residues of galactose-1-phosphate uridylyltransferase are conserved in APAT and Ap(4)A phosphorylase, suggesting a histidine as the nucleotide-binding residue in all three enzymes, which together form a new family of nucleotidyltransferases. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 2020 - 2 The subunit structure of nitrite reductase purified from the denitrifier Achromobacter cycloclastes; Inatomi K; The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution . In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit . Signal-peptide cleavage sites in its N-terminal region are discussed. Eur J Biochem, 2000 Jan, 267(2), 422 - 33 Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans; Schwarze C et al.; Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions . The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation . By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA . Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes . This reaction involves cytochrome c551 in a diffusional process . Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol . Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin . The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool . Chromatophores contain approximately 20 ubiquinone-10 molecules per RC . At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation . Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer . Results can be rationalized in the framework of a Q-cycle-type model. J Biol Inorg Chem, 1999 Dec, 4(6), 749 - 58 Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp . strain J and the structure of novel Az-iso2; Suzuki S et al.; Two azurin-type blue copper proteins, which are related to the electron-transfer processes involving methylamine/methanol oxidation, have been spectroscopically and electrochemically characterized . The obligate methylotroph Methylomonas sp . strain J gives rise to two azurins (Az-isol and Az-iso2) with methylamine dehydrogenase (MADH-Mj) . The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620-630 nm in those of usual azurins . The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character . The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry . The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively . The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5 . However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1 . The structure of Az-iso2 has been determined and refined against 1.6 A X-ray diffraction data . The whole structure of Az-iso2 is quite similar to those of azurins reported already . The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different . These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH. Arch Microbiol, 2000 Nov, 174(5), 307 - 13 Evidence for a functional similarity between the two-component regulatory systems RegSR, ActSR, and RegBA (PrrBA) in alpha-Proteobacteria; Emmerich R et al.; The symbiotic bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti, and the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodovulum sulfidophilum, Roseobacter denitrificans and Rhodobacter sphaeroides possess homologous two-component regulatory systems, namely RegSR, ActSR, RegBA and PrrBA . The respective response regulators of these bacteria control expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance . We therefore asked whether the regulators are functionally exchangeable or whether they have disparate functions in the different species, despite the amino acid sequence similarity . In this study, we showed that purified B . japonicum RegR bound in vitro to genuine DNA targets for Rba . capsulatus RegA, and that RegA was phosphorylated in vitro when RegSc (a soluble variant of the sensor kinase RegS) was added to an Escherichia coli extract containing overexpressed RegA . In vivo, RegA and S . meliloti ActR activated transcription of the B . japonicum fixR-nifA operon, normally a target for RegR . The genes for both regulators, regA and actR, were able to complement a B . japonicum regR mutant with respect to the formation of a nitrogen-fixing symbiosis with soybean . Vice versa, RegR activated in Rba . capsulatus the expression of the photosynthesis operon puc, normally a target for RegA . In conclusion, the results show that B . japonicum RegR, Rba . capsulatus RegA, and S . meliloti ActR are functionally similar. J Mol Biol, 2000 Jan 21, 295(3), 667 - 78 Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states; Harrenga A et al.; The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1 . 4 A for the reduced state . This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states . The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes . In general, the structure has the same features as that of known eukaryotic cytochromes c . However, the surface properties are very different . Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms . One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c . It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A . Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c . The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one . FEBS Lett, 1999 Dec 3, 462(3), 416 - 20 Sequence conservation from human to prokaryotes of Surf1, a protein involved in cytochrome c oxidase assembly, deficient in Leigh syndrome; Poyau A et al.; The human SURF1 gene encoding a protein involved in cytochrome c oxidase (COX) assembly, is mutated in most patients presenting Leigh syndrome associated with COX deficiency . Proteins homologous to the human Surf1 have been identified in nine eukaryotes and six prokaryotes using database alignment tools, structure prediction and/or cDNA sequencing . Their sequence comparison revealed a remarkable Surf1 conservation during evolution and put forward at least four highly conserved domains that should be essential for Surf1 function . In Paracoccus denitrificans, the Surf1 homologue is found in the quinol oxidase operon, suggesting that Surf1 is associated with a primitive quinol oxidase which belongs to the same superfamily as cytochrome oxidase. FEMS Microbiol Ecol, 2000 Jan 1, 31(1), 73 - 86 Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil; Chidthaisong A et al.; Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using {U-(14)C}glucose and {2-(14)C}acetate . The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate . Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not . These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations . Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O) . Addition of nitrate resulted in complete inhibition of CH(4) production from {U-(14)C}glucose and {2-(14)C}acetate . However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from {U-(14)C}glucose by only 70 and 65%, respectively . None of the electron acceptors significantly increased the production of (14)CO(2) from {U-(14)C}glucose, but all increased the production of (14)CO(2) from {2-(14)C}acetate . Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control . Addition of ferrihydrite and sulfate reduced (14)CH(4) production from {2-(14)C}acetate by 83 and 92%, respectively . Chloroform completely inhibited the methanogenic consumption of acetate . It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite . Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production. Biochim Biophys Acta, 2000 Jan 3, 1456(1), 1 - 4 Interaction between the formyl group of heme a and arginine 54 in cytochrome aa(3) from Paracoccus denitrificans; Riistama S et al.; The optical spectrum of heme a is red-shifted in aa(3)-type cytochrome c oxidases compared to isolated low-spin heme A model compounds . Early spectroscopic studies indicated that this may be due to hydrogen-bonding of the formyl group of heme a to an amino acid in the close vicinity . Here we show that most of the optical spectral shift of native heme a is due to a hydrogen-bonding interaction between the formyl group and arginine-54 in subunit I of cytochrome aa(3) from Paracoccus denitrificans, and that a smaller part is due to an electrostatic interaction between the D ring propionate of heme a and arginine-474. Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14718 - 23 The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications; Giuffre A et al.; We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions . The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation . Thus, contrary to the eukaryotic enzyme, both T . thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at {NO} approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant . We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction . We propose that the NO reductase activity of T . thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure . These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases . Our findings represent functional evidence in support of this hypothesis. J Biol Chem, 1999 Dec 31, 274(53), 37974 - 81 Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase; Kannt A et al.; The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential . The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O . Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species . A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3) . This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process . This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN . Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex. Mol Microbiol, 1999 Oct, 34(1), 24 - 36 Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein; Saunders NF et al.; The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively . The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of {4Fe-4S} clusters in many ferredoxin-like proteins . NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence . NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti . Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides . The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type . Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators . An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes . Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon. J Appl Microbiol, 1999 Sep, 87(3), 353 - 8 Effects of cultural conditions on denitrification by pseudomonas stutzeri measured by membrane inlet mass spectrometry Firth JR, Edwards C. Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter . Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium . MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen . A nitrate concentration of 15 mmol l-1 and a nitrite concentration of 5 mmol l-1 were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released . Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products . Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps . stutzeri is the amount of nitrite generated at the first enzymatic stage of the process . This appears to cause inhibition of the denitrification pathway above 5 mmol l-1 and at high enough concentrations (15 mmol l-1) restricts growth. Microbiology, 1999 Nov, 145 ( Pt 11), 3265 - 71 Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate; Leutwein C et al.; Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate . This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica . Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds . The authors show here by experiments with cell extracts of toluene-grown T . aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible succinyl-CoA-dependent CoA-transferase . The product of the CoA-transferase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway . The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed . Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates . In accordance, the reaction of succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(-)-enantiomer. Microbiology, 1999 Nov, 145 ( Pt 11), 3047 - 57 Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB; Page MD et al.; Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by omega insertion . Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids . All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa3 . Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not . The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant . In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT . Siderophore biosynthesis and/or secretion by P . denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD . These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG. Appl Environ Microbiol, 1999 Dec, 65(12), 5493 - 9 Partitioning effects during terminal carbon and electron flow in sediments of a low-salinity meltwater pond near Bratina Island, McMurdo Ice Shelf, Antarctica; Mountfort DO et al.; A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5 degrees C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes . Addition of {2-(14)C}acetate to sediment samples resulted in the passage of label mainly to CO(2) . Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm(-3) h(-1) compared to 2.5 to 6 nmol cm(-3) h(-1)), the portion of methane production attributed to acetate cleavage was <2% . Substantial increases in the methane production rate were observed with H(2) (2.4-fold), and H(2) uptake was totally accounted for by methane production under physiological conditions . Formate also stimulated methane production (twofold), presumably through H(2) release mediated through hydrogen lyase . Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, approximately 0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM) . No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis . The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H(2) and CO(2) where chloride, but not sulfate, has a modulating role . Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20 degrees C. Appl Environ Microbiol, 1999 Dec, 65(12), 5484 - 92 Biodegradation of free phytol by bacterial communities isolated from marine sediments under aerobic and denitrifying conditions; Rontani JF et al.; Biodegradation of (E)-phytol {3,7,11, 15-tetramethylhexadec-2(E)-en-1-ol} by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20 degrees C . This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10, 14-trimethylpentadecan-2-one and (E)-phytenic acid . The first step in both aerobic and anaerobic bacterial degradation of (E)-phytol involves the transient production of (E)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one . Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations . Since (E)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15 degrees C), the major part of (E)-phytol appeared to be biodegraded in situ via (E)-phytenic acid . (Z)- and (E)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core . This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating beta-decarboxymethylation and beta-oxidation reaction sequences induced by denitrifiers . Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (E)-phytol. Biotechnol Bioeng, 2000 Jan 5, 67(1), 19 - 24 Modeling of biological processes using self-cycling fermentation and genetic algorithms; Pinchuk RJ et al.; Self-cycling fermentation (SCF) was coupled with a genetic algorithm (GA) to provide a simple system for evaluating biological models . The SCF provided the necessary system excitation and data "richness" required to completely define the fitted biological models . The solution scheme based on the GA avoided the computational difficulties often associated with calculus-based nonlinear regression techniques, resulting in rapid and accurate convergence . After validating the mathematical approach, data from the SCF obtained under denitrifying conditions were fitted successfully to an established model using the GA . Finally, data obtained in the SCF for the removal of phenol were used to compare multiple models . This work suggests that the SCF, in conjunction with the GA, provides a coherent system that can facilitate the characterization of biological systems . Bioorg Med Chem, 1999 Oct, 7(10), 2215 - 9 Genetic engineering of Escherichia coli for the production of precorrin-3 in vivo and in vitro; Roessner CA et al.; The construction of a new recombinant strain of Escherichia coli in which two vitamin B12 biosynthetic genes, cobA and cobI, from Pseudomonas denitrificans are simultaneously overexpressed has resulted in the in vivo synthesis and accumulation of Factor III, an isobacteriochlorin not normally synthesized in E . coli . A lysate of the new strain can take the place of two lysates normally required to provide uroporphyrinogen III methyltransferase (cobA) and precorrin-2 methyltransferase (cobI) in an anaerobic five-enzyme synthesis of the early B12 intermediate, precorrin-3 (the reduced form of Factor III) from delta-aminolevulinic acid. Biochemistry, 1999 Nov 16, 38(46), 15150 - 6 Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase; Ma J et al.; Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase . Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix . The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P . denitrificans oxidase) . Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase . The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase . Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center . The data suggest that E89II (E . coli numbering) is critical for the function of the heme copper oxidases . The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel . This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant . Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants. Analyst, 1999 Feb, 124(2), 129 - 34 Optical biosensing of nitric oxide using the metalloprotein cytochrome c'; Blyth DJ et al.; The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO) . The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements . Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre . Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period . The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO . However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%) . An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1. J Biol Chem, 1999 Nov 19, 274(47), 33296 - 9 The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction; Harrenga A et al.; Cytochrome c oxidase catalyzes the reduction of oxygen to water . This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping") . The mechanism of proton pumping is still a matter of debate . Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase . Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K . No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed . The three histidine Cu(B) ligands are well defined in the oxidized and in the reduced states . These results are hardly compatible with the "histidine cycle" mechanisms formulated previously. J Bacteriol, 1999 Nov, 181(22), 6907 - 13 Anaerobic growth of Paracoccus denitrificans requires cobalamin: characterization of cobK and cobJ genes; Shearer N et al.; A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated . This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media . Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway . Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase . The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P . denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions . Comparison of the cobK and cobJ genes with their orthologues suggests that P . denitrificans uses the aerobic pathway for cobalamin synthesis . It is paradoxical that under anaerobic growth conditions, P . denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis . Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media . These observations provide evidence that P . denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions. Arch Microbiol, 1999 Nov, 172(5), 303 - 12 Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria; Harms G et al.; The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud . Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks) . Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months . Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail . Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively . Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene . Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., alpha-terpinene . In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids . Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions . Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene . Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack . Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1 . Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the beta-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis . This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes. Biochem Biophys Res Commun, 1999 Nov, 265(1), 177 - 83 The cbbQ genes, located downstream of the form I and form II RubisCO genes, affect the activity of both RubisCOs; Hayashi NR et al.; Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1 and cbbLS-2) and one form II type (cbbM) enzymes . The cbbQ-m gene was located downstream of cbbM, and showed high similarity to other cbbQ genes and nirQ/norQ genes in denitrifying bacteria . Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv . marinus closely resembled CbbQ from Thiobacillus intermedius and Thiobacillus neapolitannus and less closely resembled NirQ and NorQ . The cbbQ-m gene has been shown to activate the form II RubisCO in E . coli cells, and the cbbQ-t from Hydrogenophilus thermoluteolus could also activate the form II RubisCO . Both cbbQ genes have also been shown to activate the form I RubisCO from Hp . thermoluteolus in E . coli cells . However, the activation levels of two form I RubisCOs from Hv . marinus were smaller than that of form I RubisCOs from Hp . thermoluteolus . Form II RubisCO activated by CbbQ-m (QM) was purified from E . coli cells . The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ . Arch Microbiol, 1999 Oct, 172(4), 204 - 12 Phototrophic utilization of toluene under anoxic conditions by a new strain of blastochloris sulfoviridis Zengler K, Heider J, Rossello-Mora R, Widdel F. The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene . When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed . Agar dilution series indicated the dominance of two types of phototrophic bacteria . One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene . The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate . One strain of the latter type, ToP1, was studied in detail . Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the alpha-subclass of Proteobacteria . However, the type strain (DSM 729) of Blc . sulfoviridis grew neither on toluene nor on benzoate . Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments . Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon . In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected . These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria . The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats . Counting series revealed that up to around 1% (1.8 x 10(5) cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene. Biochim Biophys Acta, 1999 Oct 12, 1434(2), 248 - 59 A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization; Alves T et al.; Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions . The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps . N-terminal sequence comparison showed that the Ps . nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa . UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem . Monohaemic cytochrome c(552) from Ps . nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116 inverted question mark omitted inverted question mark000 min(-1) . Using this cytochrome the enzyme retained the same activity even at high ionic strength . There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. Biochim Biophys Acta, 1999 Sep 1, 1413(1), 1 - 13 The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel; Pereira MM et al.; The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283) . In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised . It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type . This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase . The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3) . Subunit I of R . marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels) . However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R . marinus oxidase . Homology modelling of the R . marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre . Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV- . These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel. J Microbiol Methods, 1999 Oct, 38(1-2), 91 - 9 Effects of storage on measurements of potential microbial activities in stream fine benthic organic matter; Bonin HL et al.; Sample storage can significantly influence measured microbial activities in stream fine benthic organic matter (FBOM), possibly confounding effects of sample variability and short-term changes in activity . Denitrification potential, acetylene reduction and respiration rates, mineralizable N and extractable ammonium concentrations, and beta-glucosidase and phosphatase enzyme activities of FBOM from first-order mountain streams in the western Oregon Cascade Mountains were assayed at various times after collection to determine potential storage effects . Denitrification potential, phosphatase activity, and extractable ammonium remained stable over a minimum of 11 h of storage at 5 degrees C . Mineralizable N concentrations, respiration rates, and beta-glucosidase activity all decreased within 12 h of collection . Results varied for acetylene reduction . Once assay conditions were established, denitrification potential and respiration rates were linear with incubation time . Based on paired t-tests, measures of acetylene reduction, denitrification potential, respiration rate, beta-glucosidase activity, and phosphatase activity were generally similar at a 1-wk interval within the same stream reaches. Carbohydr Res, 1999 Jun 30, 319(1-4), 133 - 40 Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960; Parolis LA et al.; The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy . The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments . The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot . The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds . Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid. J Bacteriol, 1999 Oct, 181(20), 6403 - 10 Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp . strain T; Krieger CJ et al.; The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp . strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate . Permeabilized cells of m-xylene-grown Azoarcus sp . strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate . In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate . Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp . strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption . Based on these findings, we propose that Azoarcus sp . strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA . A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond. Biospectroscopy, 1999, 5(5 Suppl), S19 - 32 Paramagnetic NMR studies of blue and purple copper proteins; Kolczak U et al.; 1H- and 13C-NMR spectroscopy is applied to investigate the CU(A) and type 1 active sites of copper proteins in solution . The analysis of hyperfine shifted 1H resonances allows the comparison of the electron spin density delocalization in the CU(A) site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin) . Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues . A reevaluation of the Cys H(beta) chemical shifts that is in agreement with the data published for both the P . denitrificans and the P . versutus Cu(A) soluble domains confirms the thermal accessibility of the 2B(3u) electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands . The 13C-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments . The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron. FEMS Microbiol Ecol, 1999 Oct 1, 30(2), 113 - 123 Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group; Sorokin DY et al.; A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate . The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions . Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions . In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N(2)O as electron acceptor . The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions . Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N(2)O as electron acceptors . Others functioned best with N(2)O during anaerobic thiosulfate oxidation . The latter strains appeared to have a lower level of nitrite reductase activity . Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor . The strains had DNA-DNA similarity levels higher than 30% . Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica . As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed 'Flavobacterium lutescens' (ATCC 27951) is also a P . stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N(2)O . The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P . stutzeri 'superspecies', can play a much more significant role in the biogeochemical cycles than was previously recognized. Biochim Biophys Acta, 1999 Oct 6, 1447(1), 57 - 63 Site-directed mutagenesis of the response regulator DmsR for the dmsCBA operon expression in Rhodobacter sphaeroides f . sp . Denitrificans: An essential residue of proline-130 in the linker; Yamamoto I et al.; DmsR protein is a member of the OmpR response regulator subfamily that activates the transcription of the dmsCBA operon in Rhodobacter sphaeroides f . sp . denitrificans . By site-directed mutagenesis some functional amino acid residues were investigated in DmsR, which consists of the N-terminal regulatory and the C-terminal DNA-binding domains and the linker connecting the two domains . The substitution of P130S in the linker caused decreases of both DNA-binding and transcriptional activator activities . Introducing additional substitutions of R129P or D131P to the DmsR-P130S derivative recovered both activities, demonstrating necessity of proline residue at one of the positions 129-131 in the linker . Substitutions of D12A, D55A, and K104M, at residues conserved in the phosphorylation region, caused no production of DMSO reductase, but retained DNA-binding ability, suggesting that unphosphorylated DmsR also has high affinity to its target nucleotide sequence of DNA . Substitutions in the C-terminal domain suggested the presence of a winged helix-turn-helix structure observed in the DNA-binding domain of the Escherichia coli OmpR. J Bacteriol, 1999 Oct, 181(19), 6028 - 32 Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f . sp . denitrificans IL106; Sabaty M et al.; We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f . sp . denitrificans IL106 . A mutant with this enzyme deleted is unable to grow under denitrifying conditions . Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate . Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase . This demonstrates that R . sphaeroides f . sp . denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species . In contrast, we show that nitrite induces the synthesis of the nitrate reductase. J Biol Chem, 1999 Oct 1, 274(40), 28606 - 11 H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans . Studies on topology and stoichiometry of the peripheral subunits; Yano T et al.; The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane . In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P . denitrificans NDH-1 in the membranes were investigated using immunological techniques . Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes . Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex . Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association . In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed . In addition, determination of stoichiometry of the peripheral subunits of the P . denitrificans NDH-1 was completed by radioimmunological methods . All the peripheral subunits are present as one molecule each in the enzyme complex . These results estimated the total number of cofactors in the P . denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x{2Fe-2S} and 6x{4Fe-4S}, provided that the NQO6 subunit bears one {4Fe-4S} cluster. J Biol Chem, 1999 Oct 1, 274(40), 28598 - 605 Characterization of the putative 2x{4Fe-4S}-binding NQO9 subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans . Expression, reconstitution, and EPR characterization; Yano T et al.; Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2x{4Fe-4S} clusters, were investigated using recombinant expression techniques and EPR spectroscopy . The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification . Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm . The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S(2-) (2.0-2.2 mol each per mol of subunit) . However, the iron-sulfur content was considerably increased by in vitro reconstitution . The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S(2-) per molecule and exhibited optical absorption spectra similar to those of 2x{4Fe-4S} ferredoxins . Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K) . Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g(, perpendicular) = 2.05, approximately 1.93, and g(parallel, perpendicular) = 2.08, approximately 1.90, and respective slow (P((1)/(2)) = 8 milliwatts) and fast (P((1)/(2)) = 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g(parallel, perpendicular) = 2.05, approximately 1.93) with slow spin relaxation . When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g(parallel, perpendicular) = 2.08, approximately 1.90, with enhanced spin relaxation . In either case, our EPR data provide the first experimental evidence for the presence of two {4Fe-4S} iron-sulfur clusters in the NQO9 subunit. Nature, 1999 Sep 9, 401(6749), 181 - 4 Coherent reaction dynamics in a bacterial cytochrome c oxidase; Liebl U et al.; Biological reactions in protein complexes involve structural dynamics spanning many orders of magnitude in time . In standard descriptions of catalysis by enzymes, the transition state between reactant and product is reached by thermal, stochastic motion . In the ultrashort time domain, however, the protein moiety and cofactor motions leading to altered conformations can be coherent rather than stochastic in nature . Such coherent motions may play a key role in controlling the accessibility of the transition state and explain the high efficiency of the reaction . Here we present evidence for coherent population transfer to the product state during an ultrafast reaction catalysed by a key enzyme in aerobic organisms . Using the enzyme cytochrome c oxidase aa3 from the bacterium Paracoccus denitrificans, we have studied haem dynamics during the photo-initiated ultrafast transfer of carbon monoxide from haem a3 to CuB by femtosecond spectroscopy . The ground state of the unliganded a3 species is populated in a stepwise manner in time, indicating that the reaction is mainly governed by coherent vibrations (47cm(-1)) . The reaction coordinate involves conformational relaxation of the haem group and we suggest that ligand transfer also contributes. J Clin Microbiol, 1999 Oct, 37(10), 3374 - 9 Capillary electrophoresis-single-strand conformation polymorphism analysis for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis; Ghozzi R et al.; We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF) . Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes . The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer . Data were analyzed with GeneScan 672 software . This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers . Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism . The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 {M . N . Widjojoatmodjo, A . C . Fluit, and J . Verhoef, J . Clin . Microbiol . 32:3002-3007, 1994}) . This primer set differentiated the main CF pathogens from closely related species but did not distinguish P . aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans . Two hundred seven CF clinical isolates (153 of P . aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A . xylosoxidans) were tested with P11P-P13P . The CE-SSCP patterns obtained were identical to those for the corresponding reference strains . Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains . This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF. FEMS Microbiol Lett, 1999 Sep 1, 178(1), 147 - 53 Substrate range of benzylsuccinate synthase from Azoarcus sp . strain T; Beller HR et al.; Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria . In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp . strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane) . Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate . Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer {(R)-2-benzyl-3-carboxypropionic acid} . The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates . The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed. J Bacteriol, 1999 Sep, 181(18), 5662 - 8 In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization; Johnson HA et al.; Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp . strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol . 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate . We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain . With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp . strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) {mg of protein}(-1) and an apparent K(m) for ethylbenzene of approximately 60 microM . The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene . Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis . Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone . When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase . Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp . strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells. FEBS Lett, 1999 Sep 17, 458(2), 83 - 6 Similarities and dissimilarities in the structure-function relation between the cytochrome c oxidase from bovine heart and from Paracoccus denitrificans as revealed by FT-IR difference spectroscopy; Hellwig P et al.; The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT-IR spectroscopic approach . A direct comparison to the electrochemically induced FT-IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes . These differences are partially attributed to interactions of subunits influencing the heme and protein modes . In the spectral regions characteristic for v(C=O) and v(COO-)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm-1 are observed . On this basis, the possible involvement of Asp-51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X-ray structures, is critically discussed. Appl Environ Microbiol, 1999 Sep, 65(9), 4189 - 96 On the occurrence of anoxic microniches, denitrification, and sulfate reduction in aerated activated sludge; Schramm A et al.; A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge . Microsensor measurements (O(2), NO(2)(-), NO(3)(-), and H(2)S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale . Incubations of activated sludge with (15)NO(3)(-) and (35)SO(4)(2-) were used to determine denitrification and sulfate reduction rates on a batch scale . In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low . However, in two sludges anoxia in flocs coincided with significant denitrification rates . Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions . In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase . A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels . This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges. Biophys J, 1999 Sep, 77(3), 1694 - 711 The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region; Horvath MP et al.; The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a . This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures . Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data . Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3) . Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore . The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition. Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 562 - 4 Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans; Koutny M et al.; The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S . (1997) Biochim . Biophys . Acta 1318, 202-216) does not involve the concentration of an electron donor . Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide . NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme . The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex . FEBS Lett, 1999 Aug 13, 456(3), 365 - 9 Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase; Pfitzner U et al.; Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin . We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion . Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer . Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions . This spectral behavior, largely comparable to that of the mitochondrial enzyme, was n