Journal of Bacteriology, March 2004, p . 1320-1329, Vol . 186, No . 5

Functionally Critical Elements of CooA-Related CO Sensors

Hwan Youn, Robert L . Kerby, Mary Conrad, and Gary P . Roberts^*

Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 2 October 2003/ Accepted 24 November 2003

 
CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source . We have identified a number of CooA homologs throughdatabase searches, expressed these heterologously in Escherichiacoli, and monitored their ability to respond to CO in vivo.Further in vitro analysis of two CooA homologs from Azotobactervinelandii and Carboxydothermus hydrogenoformans corroboratedthe in vivo data by revealing the ability of CO to bind to thesehemoproteins and stimulate their binding at specific DNA sequences.These data, as well as the patterns of conserved residues inthe homologs, are compared to what is already known about functionallyimportant residues in the CooA protein of R . rubrum . The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.

 
The CO-dependent anaerobic growth of Rhodospirillum rubrum relies on a CO oxidation system encoded by two CO-regulated transcriptional units, cooMKLXUH and cooFSCTJ [8, 9, 15-17, 32] . The key productsof the coo regulon are an O₂-sensitive CO dehydrogenase [CooS],a CooS-associated Fe-S protein [CooF], and a CO-tolerant hydrogenase[CooH], and the expression of the genes depends upon the activityof the CooA protein, which senses CO under anaerobic conditions.CooA, a member of the CRP/FNR [cyclic AMP receptor protein/fumaratenitrate reductase] transcriptional regulator family, is a homodimerin which each monomer contains a b-type heme [34] . Remarkably,CooA senses both the redox state of the cell and CO, since theheme undergoes reduction at approximately -300 mV [24] and only the reduced form [heme Fe[II]] of CooA binds the effector [30, 32] . An unusual switch between the Cys75 and His77 heme ligands,which is presumably important for setting the proper heme ironredox poise, accompanies the oxidation-reduction of the CooA heme [2, 33].

The crystal structure of Fe[II] CooA has been solved [Fig . 1] and provides a basis for comparing the various CooA homologs. That analysis revealed a novel subunit-swapped N-terminal Pro2as the heme ligand trans to His77 in the homodimer [20] . Nuclearmagnetic resonance and resonance Raman studies indicate that CO displaces the Pro2 ligand of Fe[II] CooA [40, 43] and exposesthe CO-bound heme to the long -helices [termed the C-helices]that extend along the homodimer interface [4, 44, 45] . Extensivemutagenic analysis suggests that CooA activation involves themonomer repositioning about the C-helices at the dimer interface[14, 44, 45] . Therefore, our present hypothesis for CooA activationposits that CO binding displaces the endogenous Pro2 ligand,which allows an interaction of the CO-bound heme with the C-helices.This interaction stabilizes an alternative domain conformationabout these helices which alters the hinge region [lying betweenC- and D-helices] that separates the DNA- and CO-binding domains[20] [Fig . 1] . The alteration of the geometry of the hinge regiondestabilizes the inactive form of CooA and stabilizes the activeform, which is presumably similar to the active form of CRP[26].

 
CO has also been suggested to serve as a neural signal in vertebrates, perhaps by interacting with soluble guanylyl cyclase, although the matter remains controversial [3] . Until this is understood,the CooA protein from R . rubrum remains the only naturally occurringCO sensor that has been proven to be physiologically relevant.

Similar CO oxidation systems have been reported for Carboxydothermus hydrogenoformans [37] and Desulfovibrio vulgaris, and a reportof a CooA homolog in the latter organism [41] motivated us toexamine the databases of completed and unfinished genomes forother CooA homologs . Eight homologs were identified in six organisms;all of these genomes also possess homologs to the CO oxidationsystem of R . rubrum, which suggests that the CooA homologs mightwell serve as CO sensors . The present work reports the functionalcharacterization of the CooA homologs, which, together withprevious work on CooA from R . rubrum, provides functional commonalitiesamong the CO sensors.

 
Sequence searches and alignment. The R . rubrum CooA protein sequence was used as the query ina TBLASTN [version 2.2.4] search of the entire genome databaseof completed and unfinished microbial sequences [207 sequences]at the National Center for Biotechnology Information [1] . Extractedsequences were aligned by using the T-COFFEE protocol [version1.37] as implemented at http://www.cmbi.kun.nl/bioinf/tools/T_COFFEE/[25].

Cloning of cooA homologs. The cooA homologs were cloned into EcoRI-HindIII-digested pEXT20[6] after genomic PCR amplification with 5' [containing EcoRI]and 3' [containing HindIII] primers designed according to each cooA sequence with previously described extensions [14] . Thecloned homologs included Azotobacter vinelandii CooA, C . hydrogenoformansCooA [C . hydrogenoformans 2350 CooA], and D . vulgaris HildenboroughCooA . For Desulfovibrio desulfuricans G20 CooA and Desulfitobacteriumhafniense CooA, an EcoRI site inside the cooA gene was eliminated [see below] without changing the protein sequence before cloningthe PCR product into the EcoRI/HindIII-digested vector . C . hydrogenoformansalso contained a second cooA [C . hydrogenoformans 2340 CooA]as well as a second cluster of genes homologous to the coo genesof R . rubrum [37] . This cooA also contained an internal EcoRIsite and was therefore cloned as a blunt-ended [5'] and HindIII-digested [3'] PCR fragment into SmaI/HindIII-digested pEXT20 . Genomic DNAs were kindly supplied by Luis M . Rubio, University of California, Berkeley [A . vinelandii]; Frank Robb, University of Maryland, Baltimore [C . hydrogenoformans]; Gerrit Voordouw, University of Calgary, Calgary, Alberta, Canada [D . vulgaris Hildenborough]; Judy Wall, University of Missouri—Columbia, Columbia [D. desulfuricans G20]; and Richard Villemur, INRS-Institut Armand-Frappier, Laval, Quebec, Canada [D . hafniense strain DCB-2] . When necessary, mutations were introduced into the cooA genes by the QuikChange procedure [Stratagene, La Jolla, Calif.].

Reporter system for CooA transcription activity in vivo. The in vivo activities of the CooA homologs were measured bytheir abilities to stimulate ß-galactosidase synthesiswith the E . coli reporter strain developed for the analysisof CooA of R . rubrum, which has been described previously [33]. Anaerobic expression utilized 120-ml stoppered serum vials containing 20 ml of morpholinepropanesulfonic acid [MOPS]-buffered medium[38] supplemented with 100 µg of ampicillin [Na⁺ salt]/mland 25 µM isopropyl-ß-D-thiogalactopyranoside [IPTG] . For CO-induced cultures, the headspace of the vialscontained 2% [vol/vol] CO gas . Cultures were grown with shakingat 30°C to an optical density at 600 nm of 1.5 to 1.8, cellpellets were prepared and frozen, and ß-galactosidaseactivities were measured by a standard protocol [28].

CooA homolog expression and purification. For the partial purification of certain CooA proteins, the expressionvectors were transferred into host strain VJS6737, which wasa gift of Valley Stewart [35] . For unknown reasons, the levelof CooAs expressed in this strain often exceeds those of ourstandard reporter-containing host by 2- to 10-fold . Strainswere cultivated at 30°C in 2x LC medium [14] supplementedwith phosphate buffer [pH 7.0] to 10 mM, glucose to 10 mM, ampicillin[Na⁺ salt] to 100 µg/ml, and IPTG to 500 µM . Aerobiccultures involved the use of 40 ml of medium in 250-ml flasksagitated at 250 rpm; anaerobic cultures employed 200 ml of mediumin 250-ml screw-cap bottles that were gently mixed at 80 rpm. R . rubrum CooA, A . vinelandii CooA, and C . hydrogenoformans2340 CooA homologs were partially purified with a batch hydroxylapatitemethod as described previously [45], as spectral analysis showedthat R . rubrum CooA and C . hydrogenoformans 2340 CooA were stableduring aerobic purification [data not shown] . The heme contentof the CooA preparations was estimated by a modified reduced pyridine-hemochromogen method [42], and protein concentration was measured by the bicinchoninic acid assay [Pierce, Rockford, Ill.] . UV-visible absorption spectroscopy of CooA samples in25 mM MOPS buffer, pH 7.4, with 0.1 M NaCl, was performed atroom temperature in quartz cuvettes with a Shimadzu UV-2401PC spectrophotometer . Samples were made anaerobic by flushing withargon and were reduced by the addition of an anaerobically prepared dithionite solution [final concentration, 2 mM] . Anaerobic COgas [final concentration, 20% [vol/vol]] was further added andmixed by gentle inversion for the CO-bound spectra . Potassiumferricyanide [20 µM final concentration] was used forthe oxidation of isolated C . hydrogenoformans 2340 CooA.

In vitro DNA-binding assay. In vitro DNA-binding assays of CooA homologs were performedby using the fluorescence polarization technique with a Beacon2000 fluorescence polarization detector [PanVera Corp., Madison,Wis.] as described previously [22, 39] . As a fluorescence probe,a 26-bp target DNA containing R . rubrum P_cooF was labeled with Texas Red on one end of the duplex and used at a concentrationof 6.4 nM . Binding assays were performed in 40 mM Tris-HCl [pH8.0], 6 mM CaCl₂, 50 mM KCl, 5% [vol/vol] glycerol, and 1 mM dithiothreitol with salmon sperm DNA added at 6.4 µM asa nonspecific competitor . The conditions for reduction and COtreatment of the samples were similar to those used for obtainingUV-visible spectra.

 
Discovery and identification of CooA homologs. The report by Voordouw [41] of a gene encoding a CooA homologin D . vulgaris prompted us to search the database for other cooA homologs . Eight genes from six organisms were identified [Table 1 and Fig . 2] that appeared to encode CO sensors basedon the following criteria . [i] They showed substantial proteinsequence similarity, ranging from 49 to 55%, compared to R.rubrum CooA . [ii] The homologs were found in genomes that alsocontained genes for CO oxidation systems homologous to thatof R . rubrum . At least C . hydrogenoformans and D . vulgaris havealso been shown to have CO dehydrogenase activity [5, 41] . [iii]Near the invariant histidine proximal ligand, all homologs containedan internal deletion of eight or nine codons [with respect tothe CRP] that appears to provide space for the heme in CooAof R . rubrum . [iv] The alignment showed significant conservationof R . rubrum CooA residues known to be critical for proteinfunction [discussed below] . Notably absent from the list areorganisms wherein CO oxidation is an aerobic process catalyzedby a molybdenum-containing hydroxylase [23] as well as anaerobicorganisms [e.g., methanogenic archaea] in which the capabilityof CO oxidation reflects an intrinsic function of a constitutivelyexpressed metabolic process [7].

 
C . hydrogenoformans and D . hafniense each contained two cooA homologs, consistent with the observation that each contains two clusters of other coo gene homologs as well . We cloned both of the CooA homologs from C . hydrogenoformans [designated C. hydrogenoformans 2340 and 2350 CooA] because they differ at a unique cysteine residue that serves as an Fe[III] heme ligandin R . rubrum CooA [Cys75 in R . rubrum CooA] [Fig . 2] but arbitrarilycloned only a single CooA homolog from D . hafniense [Fig . 2].The genes encoding D . hafniense CooA and C . hydrogenoformans2340 CooA have the additional complication of more than onepotential start codon, an issue of particular relevance forR . rubrum CooA, since the N-terminal Pro2 of one monomer isa heme ligand in the other . The cloned region of D . hafnienseCooA was chosen in accordance with the terminus predicted bythe ORF Finder utility [http://www.ncbi.nlm.nih.gov/gorf/gorf.html and its position homologous to the R . rubrum CooA terminus [Fig. 2] . We created two clones for C . hydrogenoformans 2340 CooA,with start sites at residues -1 and -5 relative to the R . rubrumCooA sequence [Fig . 2] . As shown below, both of these clonesdisplayed substantial CooA activity, so the issue of the properterminus remains unresolved.

CO-responsive in vivo activities of CooA homologs. To determine if the CooA homologs function as CO sensors, weintroduced each cloned cooA into the Escherichia coli reporter strain we employed for the analysis of R . rubrum CooA . This system has a CooA-binding site and a promoter cloned from R. rubrum placed in front of lacZ . We reasoned that since the DNA-bindingF-helices are highly conserved among the CooA homologs [Fig.2], a CO-dependent response would affect binding of the appropriatesequence in the reporter and, given appropriate levels of CooAaccumulation and proper interaction with E . coli RNA polymerase,result in ß-galactosidase accumulation . Indeed, mostof the homologs displayed a significantly higher level of lacZexpression under anaerobic conditions in the presence of COthan in its absence, strongly suggestive that these cloned CooAhomologs are capable of serving as CO sensors in vivo [Table1] . As mentioned before, both versions of C . hydrogenoformans2340 CooA, which presumably differ in the length and identityof their N termini, displayed similar high activity . The onlyhomologs that failed to show a CO response were C . hydrogenoformans2350 CooA and D . vulgaris CooA . The former of these accumulatedvery poorly, but the basis of failure of the latter to showactivity is unclear [Table 1 and Fig . 3] . In the case of R.rubrum CooA, accumulation of heme-containing protein is revealedby a distinct reddish color of the cell pellet [Table 1] . While D . desulfuricans CooA and D . hafniense CooA appeared to accumulate poorly [Table 1 and Fig . 3], they were active in response toCO in vivo [Table 1].

 
Although a positive response in this assay clearly implies the presence of a CO-sensing protein, comparisons of the magnitudeof the response are more difficult to interpret because theassay reflects the combination of factors: the expression ofCO-responsive heme-containing protein, the fraction of thatpopulation that exists in the proper form to bind DNA, interactionswith a heterologous RNA polymerase, and the affinity of theCooA homolog for the R . rubrum P_cooF promoter . Despite the complexity,we currently believe that many of the other homologs interact substantially better with E . coli RNA polymerase than does R. rubrum CooA, based on both the in vivo results and on in vitro analyses described below . The regions of CooA that interact with RNA polymerase are termed activating regions [AR], basedon the large body of work done with CRP and FNR, another distantlyrelated homolog [18, 19] . This apparently better AR surfacein the homologs was initially surprising, since R . rubrum CooAhas been shown to function with the E . coli RNA polymerase invivo and in vitro [12, 21] . Nevertheless, there is no reasonto suppose that R . rubrum CooA would interact any better withthe heterologous RNA polymerase from E . coli than would anyother homolog . Indeed, it is interesting that R . rubrum CooA variants with improved AR interactions were easily obtainedin a screen for improved function [21], consistent with the notion that wild-type R . rubrum CooA might actually be rather poor in this regard . Based on this logic, we believe that A. vinelandii, C . hydrogenoformans 2340, and D . hafniense CooA possess enhanced AR surfaces [for interaction with RNA polymerase from E . coli] because the activity with CO is higher than that of R . rubrum CooA . This might also explain some of the activity without CO.

These results demonstrate that the majority of these homologshave a clear responsiveness to the presence of CO, consistentwith the hypothesis that they serve as CO sensors in the organismsin which they are normally found . It is therefore appropriateto compare their properties with those of R . rubrum CooA toelucidate the necessary features of the generalized CooA family.

The CooA homologs accumulate poorly under aerobic growth conditions. A preliminary experiment showed that the expressed CooA homologs accumulated heme-containing protein much less effectively thandid R . rubrum CooA in aerobically grown cells [data not shown].We felt that this poor accumulation might reflect poor stabilityof the heme during aerobic growth because all but one of thesehomologs lacked a residue homologous to Cys75 [Fig . 2], one of the Fe[III] heme ligands in R . rubrum CooA [29], and it hasalready been shown that a C75S variant of R . rubrum CooA isunstable under aerobic conditions [33].

To determine if growth conditions perturb the level of heme-containing CooA homologs in E . coli, we prepared both aerobic and anaerobic cultures producing R . rubrum, A . vinelandii, and C . hydrogenoformans2340 CooAs and compared their heme contents normalized to totalextract protein . Consistent with the hypothesis of poorer accumulationof A . vinelandii and C . hydrogenoformans 2340 CooAs under aerobicconditions, anaerobic growth provided much higher heme accumulationfor these homologs than for R . rubrum CooA [Table 2] . However,these CooA homologs were stable during and after purificationunder aerobic conditions, suggesting that the higher yield ofheme-containing protein from anaerobic expression might be relatedto external factors such as protease activity or better hemeincorporation during anaerobic protein synthesis . A plausibleexplanation for this phenomenon is that under physiologicalconditions, the A . vinelandii and C . hydrogenoformans 2340 CooAsare never exposed to oxidizing conditions in their native hosts.While A . vinelandii is an aerobe, its internal milieu is sufficiently reducing to permit operation of strictly anaerobic nitrogenase systems [27], and C . hydrogenoformans is an obligate anaerobeisolated from a hydrothermal vent [36].

 
Spectral comparison of R . rubrum, A . vinelandii, and C . hydrogenoformans 2340 CooAs reveals differences in Fe[III] and Fe[II] forms. UV-visible spectra and in vitro DNA-binding activities wereanalyzed for R . rubrum, A . vinelandii, and C . hydrogenoformans 2340 CooAs that had been partially purified from anaerobically grown cells . The C . hydrogenoformans 2340 CooA was chosen in part because it is the only homolog that has a Cys residue ata position homologous to Cys75 of R . rubrum CooA, which serves as one of the heme ligands in the six-coordinate low-spin Fe[III] form [Fig . 2 and 4A] . We were therefore interested to know whetherC . hydrogenoformans 2340 CooA showed similar spectral propertiesto those of R . rubrum CooA.

 
Figure 4B presents the UV-visible spectra of the C . hydrogenoformans2340 CooA in the "as isolated" form [expected to be Fe[III]]as well as the Fe[II], Fe[II]-CO, and chemically oxidized Fe[III]forms . Not surprisingly, CO addition to the Fe[II] form resultedin a spectral change indicative of CO binding . Overall spectralfeatures of the CO-bound form are very similar to those of CO-boundR . rubrum CooA, presumably reflecting the invariant His77 thatserves as the proximal ligand in that form of R . rubrum CooA[Fig . 2] . In contrast, the Soret peak of C . hydrogenoformans2340 CooA shows a reduced intensity ratio of the Fe[II] Soretband to the Fe[II]-CO Soret band, implying that the Fe[II] formin this homolog is altered relative to that of R . rubrum CooA.This spectral property is also a characteristic of Fe[II] P3R4 R . rubrum CooA [46], in which the Pro2 ligand is perturbed bythe deletion of the two penultimate residues, and suggests aweakened endogenous ligand trans to the invariant His in theC . hydrogenoformans 2340 CooA . Surprisingly, even aerobic purificationyielded partially reduced C . hydrogenoformans 2340 CooA, indicatedby sharply resolved and ß peaks in the spectrum ofthe as isolated form [Fig. 4B] . These and ß peakswere eliminated by treatment with the oxidant potassium ferricyanide[Fig . 4B], confirming the partially reduced state of the asisolated protein . The as isolated R . rubrum CooA did not showany spectral difference with or without 20 µM potassiumferricyanide [data not shown] . The redox potential of R . rubrumCooA has been determined to be approximately -300 mV, the thresholdbelow which the CO oxidation catalytic enzyme, CO dehydrogenase,is active . This result suggests that C . hydrogenoformans 2340CooA is shifted in its redox poise, presumably because of aless favorable Fe[III] state in this protein . The potassiumferricyanide-treated Fe[III] form of C . hydrogenoformans 2340CooA appears to predominantly be the six-coordinate form [Fig.4B] and not significantly different from that of R . rubrum CooA,but the natures of the endogenous ligands are yet to be determined.

To test whether or not Cys80 [Cys75 in R . rubrum CooA] serves as one ligand in the Fe[III] form of C . hydrogenoformans 2340 CooA, we introduced Ser at that position by site-directed mutagenesis. Both the heme accumulation and spectra of C80S C . hydrogenoformans 2340 CooA are remarkably similar to those of wild-type C . hydrogenoformans2340 CooA [Fig . 4B and C], in contrast to the low heme accumulationand Fe[III] spectral perturbation of the R . rubrum CooA C75Svariant [33] . This indicates that Cys80 is not the ligand inthe Fe[III] C . hydrogenoformans 2340 CooA . The most probablecandidate for the proximal Fe[III] heme ligand in C . hydrogenoformans2340 CooA is His82, which is homologous to the critical His77of R . rubrum CooA.

A . vinelandii CooA was also analyzed spectrally because it represented a CooA homolog containing Ser at the position homologous to Cys75 in R . rubrum CooA [Fig . 2] . Like C . hydrogenoformans 2340CooA, A . vinelandii CooA displayed a typical Fe[II]-CO spectrum,but the spectra of its Fe[II] and as isolated forms were notablydifferent from those of R . rubrum CooA [Fig . 4D] and are consistentwith a significant fraction of five-coordinate heme ligation.As for C . hydrogenoformans 2340 CooA, we presume this reflectsa weak endogenous ligand trans to the invariant His.

R . rubrum and C . hydrogenoformans 2340 CooA are the only two wild-type homologs that have Cys at the position homologousto Cys75 in R . rubrum CooA [Fig . 2], and this correlates withtheir excellent accumulation of heme-containing protein [Table 1; Fig . 3] . However, the ability of C80S C . hydrogenoformans2340 CooA to also accumulate heme well suggests that this isnot a causal relationship . This notion was further tested bythe creation and analysis of S77C A . vinelandii CooA . Neitherprotein accumulation nor spectra were significantly differentfrom that of wild-type A . vinelandii CooA [data not shown].The above results suggest that the presence of Cys at this positionis not correlated with the accumulation of heme-containing proteinunder these conditions.

As noted in the introduction, R . rubrum CooA undergoes a highly unusual ligand switch during oxidation and reduction and maintains a six-coordinate heme ligation under all conditions . One implication of the rather different redox behavior of the other homologs and the general absence in them of a strong ligand residue homologous to Cys75 [C . hydrogenoformans 2340 CooA contains a Cys residue, but it does not appear to serve as an Fe[III] ligand] is that these proteins probably do not undergo a similar redox change.Though this notion has not been experimentally tested, it isnevertheless consistent with the fact that R . rubrum is theonly one of these organisms likely to face such a physiologicaldecision.

The conclusion from these results is that the examined CooA homologs bind CO to create a species that is spectrally verysimilar to that of R . rubrum CooA . However, the homologs displaysome clear differences in the spectra of the Fe[II] and Fe[III]forms from that of R . rubrum CooA, though the molecular basisfor these differences is unknown . The results suggest that itis not merely the presence or absence of a Cys75 analog thatunderlies these differences . Rather, we assume that these otherhomologs lack other residues in the heme vicinity that affectthe properties of the Fe[II] and Fe[III] forms.

R . rubrum, A . vinelandii, and C . hydrogenoformans 2340 CooAs bind target DNA in a CO-dependent manner. The DNA-binding properties of the partially purified CooA homologswere tested by a fluorescence polarization assay [39] . This technique measures the ability of CooA to bind a known CooAbinding site that is identical to that in the in vivo assaybut without the complication of RNA polymerase interactions.As shown in Fig. 5, these CooA proteins showed substantial CO responsiveness regardless of whether the assay was normalizedto sample heme or protein content, although the lower fractionof heme-containing CooA in the A . vinelandii CooA preparation reduces its activity in the latter analysis . The observationthat these homologs display DNA-binding activity similar tothat of R . rubrum CooA indicates that their very high in vivoactivities [Table 1] reflect heterologous RNA polymerase interactions superior to those of R . rubrum CooA, insofar as the in vivo and in vitro assays depend on the same R . rubrum CooA DNA-binding sequences . Of course, conclusions cannot be extrapolated for relative affinities of these homologs for their native bindingsites in their natural hosts . These results confirm the CO responsiveness of the tested homologs in terms of DNA binding, consistent witha CO-induced conformational change to reposition the F-helices,as envisioned for R . rubrum CooA [14].

 
Sequence analysis of the regions 5' to the coo structural genes in all the organisms from which cooA genes were isolated revealed reasonably positioned binding sites corresponding to the TGTC[A/G]N₆[C/T]GACA consensus found in R . rubrum: 5' of cooS in D . desulfuricans,5' of cooF in A . vinelandii, 5' of cooF in D . hafniense, 5'of cooS in D . vulgaris . Similar sequences are found in the 5'regions of both cooF genes in C . hydrogenoformans. However,these sequences have not been confirmed experimentally as bindingsites, nor is the R . rubrum CooA consensus a robust one, asit is based on the only two known binding sites in the genome[11] . Nevertheless, examination of the F-helices of all thehomologs [Fig. 2] reveals significant similarity with one another, consistent with the notion that they bind similar sequences.For example, they all have an Arg residue homologous to Arg180of CRP that contacts the first G of the binding half-site [TGTGA]and they all have Gln at the position homologous to Glu181 ofCRP . Because this Glu181 interacts with the second G of theCRP half-site [TGTGA], this replacement in the CooA homologssuggests that the Gln might interact with the C of the CooAhalf-site [TGTC[A/G]].

Critical residues in heme region of CooA. The heme-binding region of CooA does not resemble those of othercommon heme regions, such as the PAS domain [10] or the hemedomain found in globins [13] . However, a comparison of the various CooA homologs, together with a substantial amount of mutational analysis of R . rubrum CooA, has revealed a number of critical residues in the vicinity of the heme.

All the homologs conserve a proximal heme environment [Fig. 2] that consists of an invariant His at position 77 [R . rubrumCooA numbering], small residues at position 75 and 78, and aPhe or Tyr at position 74 . As already mentioned, Cys75 is a ligand for the Fe[III] heme in R . rubrum CooA [29] and its absencein most of the homologs is consistent with the strict anaerobicphysiology of these organisms, whether a result of their environmentor metabolic function . A C75S variant of R . rubrum CooA accumulatesstable, active protein when expressed and analyzed under anaerobicconditions [33], consistent with the data for the homologs whichall have Cys or Ser residues [32], while larger residues atthis position preclude activity.

All the homologs possess a His residue homologous to His77 of R . rubrum CooA . The His77 residue of R . rubrum CooA serves as the proximal ligand in the Fe[II] and Fe[II]-CO forms and is critical for proper CO activation [33] . We assume that its roleis twofold . First, it must provide sufficient ligand strength to preclude its displacement by various small-molecule ligands, including CO, which itself must displace the trans ligand [43]. Secondly, because it remains tethered to the CO-bound heme,it helps define the position that the heme can assume . As CooA activation depends on an interaction of the CO-bound heme withthe C-helices, heme positioning is almost certainly criticalfor proper activation.

One of the striking observations from the Fe[II] structure of R . rubrum CooA was the evidence of Pro2 as the heme ligand trans to His77 [20], and indirect evidence also strongly suggeststhat it is the ligand in the Fe[III] form as well [24, 39, 45].An important role of this residue appears to be to maintainR . rubrum CooA in an inactive [non-DNA-binding] form in theabsence of CO, and it also appears to provide at least one levelof effector specificity, since other ligands such as CN^- andimidazole are unable to displace it . However, it has littleimportance in the conversion to the active form when CO is present[39] . Its dispensability is supported by the lack of conservationin the N-terminal region in the homologs [Fig . 2], where only one protein [D . hafniense CooA] potentially provides an N-terminal Pro ligand . The ligand trans to the His77 homologous position in the other CooAs is unknown, but it may be the terminal Met. In contrast to R . rubrum CooA, the large residues at position 2 should prevent the posttranslational processing that yieldsPro2 as the R . rubrum CooA terminus . The various CooA homologsalso differ in the apparent length of the N terminus, whichis again consistent with mutational data for R . rubrum CooA,where increasing or shortening the N terminus yielded variantswith substantial CO-responsive activity [39] . Because of its reasonable level of accumulation, C . hydrogenoformans 2340 CooA was tested for imidazole and CN^- binding but appeared to bind neither at concentrations up to 50 mM . This implies that the amino-terminal ligand of this homolog, at least, provides aroughly comparable type of exclusion to the binding of impropereffectors, as does Pro2 of R . rubrum CooA.

In contrast to the case of Pro2, the C-helix residues Leu116and Gly117 [Fig . 2] that form the distal heme pocket in CO-bound state of R . rubrum CooA are particularly critical for its response to CO [44, 45] . Their conservation in all the homologs suggeststhat these residues are crucial for the CO response of the entireCooA family . Recent evidence also suggests that Leu120 is alsoimportant for this CO response [R . L . Kerby and G . P . Roberts,unpublished data], and this residue is again absolutely conservedin the CooA family.

Conservation of a signal transduction pathway within CooA. The C-helices of the CRP/FNR family of regulators provide astable hydrophobic dimer interface, but at the same time, theymust allow sufficient mobility to permit an altered conformation,in at least CRP and CooA [14, 20, 26] . This has been directlyshown to be a critical signal pathway for CO binding [14] inR . rubrum CooA . It was therefore of great interest to see thatthe CooA homologs share with CRP the leucine zipper residuesIle113, Leu116, Leu120, Ile127, and Leu130 but invariably lacka typical d position residue at Cys123 [R . rubrum CooA numbering][Fig . 2] . Several residues around the interdomain hinge regionare also conserved in both the CooA family and in CRP; theseinclude Phe132, Asp134, Arg138, and Ala140 of R . rubrum CooA[Fig . 2] . This extensive conservation suggests that the C-helixrepositioning upon effector binding, as well as the mechanismfor reorienting the DNA-binding domains, may be fundamentallysimilar in all these proteins, although this hypothesis mustbe experimentally tested.

The unusual C terminus of R . rubrum CooA is not conserved in the CooA homologs. R . rubrum CooA not only possessed a unique N-terminal heme ligandbut also contained a novel run of C-terminal Asp residues [Fig.2] whose conformation, unfortunately, is unresolved in the structure[20] . The deletion or substitution of these residues generallyhas little effect on CooA activity [H . Youn and G . P . Roberts,unpublished data], and their relative unimportance is supportedby the absence of such residues in any of the homologs.

Surprising conservation of the 4/5 loop. The 4/5 loop refers to the structure formed by the ß-4and ß-5 sheets [Fig . 1] that extends from the effector-bindingdomain toward the DNA-binding domain, making contacts with thisdomain in the active form of CRP . In CRP, the tip of this loophas been shown to interact with the sigma subunit of RNA polymeraseand is termed AR3 [31], but the 4/5 loop residues immediatelypreceding and subsequent to the AR3 region are not known toprovide structural or functional importance . Surprisingly, inspectionof the sequence alignments shows the ß-4/5 loop tobe highly conserved in the CooA homologs and notably differentfrom CRP through the presence of two basic residues [Fig . 2].Indeed, the purified R51C R53C CooA double variant had low hemecontent and extremely poor DNA binding in response to CO [M.V . Thorsteinsson and G . P . Roberts, unpublished data] . Giventhe structural connection of this region to the His77 region,the proximal heme ligand in the CO-bound form, the ß-4/5 loop region containing these residues might therefore constitute a separate CO signal conduit within CooA in addition to the C-helix mechanism.

The results of the sequence comparison and functional and spectral analysis of the CooA homologs not only verifies their capabilityas CO sensors but have also corroborated the identificationof critical features of CooA that had previously only been basedon mutational and biochemical analysis of R . rubrum CooA . Thesefeatures include the His ligand of the heme, the hydrophobicdistal heme pocket, the negligible role of C-terminal poly-Asptail in the CO-sensing function of R . rubrum CooA, the modifiedleucine zipper formed by the C-helices, residues in the hingeregion between the effector- and DNA-binding domains, and thesurprising conservation of the ß-4 region . It is probablyof significance that the residues in the C-helix and the hingeregion are well conserved in CRP, suggesting a common pathwayof activation that remains to be completely elucidated . An importantdifference between R . rubrum CooA and the homologs was the lackof redox-based ligand switch in the latter class, and it isclear that a Cys75 analog is necessary but not sufficient forthis ability . Another interesting difference is in the N terminus,since it provides a heme ligand in R . rubrum CooA that servesas a factor in small-molecule specificity . It remains to beseen whether the CooA homologs have similar ligand specificities,and if so, what ligand supports this . The results provide afairly clear idea of the functionally critical elements of theCooA family and, in most cases, the biochemical property that underlies the role of those elements.

 
This work was supported by the College of Agricultural and Life Sciences at the University of Wisconsin—Madison, Madison,National Institutes of Health Grant GM53228 [to G.P.R.].

We thank Luis Rubio, Frank Robb, Gerrit Voordouw, Judy Wall,and Richard Villemur for the generous provision of genomic DNAfor the organisms that we examined.

 
* Corresponding author . Mailing address: Department of Bacteriology, University of Wisconsin—Madison, 420 Henry Mall, Madison, WI 53706 . Phone: [608] 262-3567 . Fax: [608] 262-9865 . E-mail: groberts@bact.wisc.edu.

1. Altschul, S . F., T . L . Madden, A . A . Schäffer, J . Zhang, Z . Zhang, W . Miller, and D . J . Lipman. 1997 . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Res . 25:3389-3402 .
2. Aono, S., K . Ohkubo, T . Matsuo, and H . Nakajima. 1998 . Redox-controlled ligand exchange of the heme in the CO-sensing transcriptional activator CooA . J . Biol . Chem . 273:25757-25764 .
3. Boehning, D., and S . H . Snyder. 2003 . Novel neural modulators . Annu . Rev . Neurosci . 26:105-131.
4. Coyle, C . M., M . Puranik, H . Youn, S . B . Nielsen, R . D . Williams, R . L . Kerby, G . P . Roberts, and T . G . Spiro. 2003 . Activation mechanism of the CO sensor CooA: mutational and resonance Raman spectroscopic studies . J . Biol . Chem . 278:35384-35393 .
5. Dobbek, H., V . Svetlitchnyi, L . Gremer, R . Huber, and O . Meyer. 2001 . Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster . Science 293:1281-1285 .
6. Dykxhoorn, D . M., R . St . Pierre, and T . Linn. 1996 . A set of compatible tac promoter expression vectors . Gene 177:133-136.
7. Ferry, J . G. 1999 . Enzymology of one-carbon metabolism in methanogenic pathways . FEMS Microbiol . Rev . 23:13-38.
8. Fox, J . D., R . L . Kerby, G . P . Roberts, and P . W . Ludden. 1996 . Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme . J . Bacteriol . 178:1515-1524.
9. Fox, J . D., Y . He, D . Shelver, G . P . Roberts, and P . W . Ludden. 1996 . Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. J . Bacteriol . 178:6200-6208.
10. Gong, W., B . Hao, S . S . Mansy, G . Gonzalez, M . A . Gilles-Gonzalez, and M . K . Chan. 1998 . Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction . Proc . Natl . Acad . Sci . USA 95:15177-15182 .
11. He, Y., D . Shelver, R . L . Kerby, and G . P . Roberts. 1996 . Characterization of a CO-responsive transcriptional activator from Rhodospirillum rubrum. J . Biol . Chem . 271:120-123 .
12. He, Y., T . Gaal, R . Karls, T . J . Donohue, R . L . Gourse, and G . P . Roberts. 1999 . Transcription activation by CooA, the CO-sensing factor from Rhodospirillum rubrum. The interaction between CooA and the C-terminal domain of the subunit of RNA polymerase . J . Biol . Chem . 274:10840-10845 .
13. Hou, S., T . Freitas, R . W . Larsen, M . Piatibratov, V . Sivozhelezov, A . Yamamoto, E . A . Meleshkevitch, M . Zimmer, G . W . Ordal, and M . Alam. 2001 . Globin-coupled sensors: a class of heme-containing sensors in Archaea and Bacteria . Proc . Natl . Acad . Sci . USA 98:9353-9358 .
14. Kerby, R . L., H . Youn, M . V . Thorsteinsson, and G . P . Roberts. 2003 . Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains . J . Mol . Biol . 325:809-823.
15. Kerby, R . L., P . W . Ludden, and G . P . Roberts. 1995 . Carbon monoxide-dependent growth of Rhodospirillum rubrum. J . Bacteriol . 177:2241-2244.
16. Kerby, R . L., P . W . Ludden, and G . P . Roberts. 1997 . In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ. J . Bacteriol . 179:2259-2266.
17. Kerby, R . L., S . S . Hong, S . A . Ensign, L . J . Coppoc, P . W . Ludden, and G . P . Roberts. 1992 . Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system . J . Bacteriol . 174:5284-5294.
18. Kolb, A., S . Busby, H . Buc, G . Garges, and S . Adhya. 1993 . Transcriptional regulation by cAMP and its receptor protein . Annu . Rev . Biochem . 62:749-795.
19. Lamberg, K . E., and P . J . Kiley. 2000 . FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3 . Mol . Microbiol . 38:1-12.
20. Lanzilotta, W . N., D . J . Schuller, M . V . Thorsteinsson, R . L . Kerby, G . P . Roberts, and T . L . Poulos. 2000 . Structure of the CO sensing transcription activator CooA . Nat . Struct . Biol . 7:876-880.
21. Leduc, J., M . V . Thorsteinsson, T . Gaal, and G . P . Roberts. 2001 . Mapping CooAXRNA polymerase interactions . Identification of activating regions 2 and 3 in CooA, the CO-sensing transcriptional activator . J . Biol . Chem . 276:39968-39973 .
22. Lundblad, J . R., M . Laurance, and R . H . Goodman. 1996 . Fluorescence polarization analysis of protein-DNA and protein-protein interactions . Mol . Endocrinol . 10:607-612.
23. Meyer, O., L . Gremer, R . Ferner, M . Ferner, H . Dobbek, M . Gnida, W . Meyer-Klaucke, and R . Huber. 2000 . The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase . Biol . Chem . 381:865-876.
24. Nakajima, H., Y . Honma, T . Tawara, T . Kato, S.-Y . Park, H . Miyatake, Y . Shiro, and S . Aono. 2001 . Redox properties and coordination structure of the heme in the CO-sensing transcriptional activator CooA . J . Biol . Chem . 276:7055-7061 .
25. Notredame, C., D . G . Higgins, and J . Heringa. 2000 . T-Coffee: a novel method for fast and accurate multiple sequence alignment . J . Mol . Biol . 302:205-217.
26. Passner, J . M., S . C . Schultz, and T . A . Steitz. 2000 . Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 Å resolution . J . Mol . Biol . 304:847-859.
27. Poole, R . K., and S . Hill. 1997 . Respiratory protection of nitrogenase activity in Azotobacter vinelandii-roles of the terminal oxidases . Biosci . Rep . 17:303-317.
28. Reynolds, A., and V . Lundblad. 1995 . Assay for ß-galactosidase in liquid culture, p . 13-30 . In F . M . Ausubel, R . Brent, R . E . Kingston, D . D . Moore, J . G . Seidman, J . A . Smith, and K . Struhl [ed.], Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology, 3rd ed . John Wiley & Sons, Inc., New York, N.Y.
29. Reynolds, M . F., D . Shelver, R . L . Kerby, R . B . Parks, G . P . Roberts, and J . N . Burstyn. 1998 . EPR and electronic absorption spectroscopies of the CO-sensing CooA protein reveal a cysteine-ligated low-spin ferric heme . J . Am . Chem . Soc . 120:9080-9081.
30. Reynolds, M . F., R . B . Parks, J . N . Burstyn, D . Shelver, M . V . Thorsteinsson, R . L . Kerby, G . P . Roberts, K . M . Vogel, and T . G . Spiro. 2000 . Electronic absorption, EPR, and resonance Raman spectroscopy of CooA, a CO-sensing transcription activator from R . rubrum, reveals a five-coordinate NO-heme . Biochemistry 39:388-396.
31. Rhodius, V . A., and S . J . Busby. 2000 . Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3 . J . Mol . Biol . 299:295-310.
32. Roberts, G . P., M . V . Thorsteinsson, R . L . Kerby, W . N . Lanzilotta, and T . L . Poulos. 2001 . CooA: a heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state . Prog . Nucleic Acid Res . Mol . Biol . 67:35-63.
33. Shelver, D., M . V . Thorsteinsson, R . L . Kerby, S.-Y . Chung, G . P . Roberts, M . F . Reynolds, R . B . Parks, and J . N . Burstyn. 1999 . Identification of two important heme site residues [cysteine 75 and histidine 77] in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum. Biochemistry 38:2669-2678.
34. Shelver, D., R . L . Kerby, Y . He, and G . P . Roberts. 1997 . CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein . Proc . Natl . Acad . Sci . USA 94:11216-11220 .
35. Stewart, V., Y . Lu, and A . J . Darwin. 2002 . Periplasmic nitrate reductase [NapABC enzyme] supports anaerobic respiration by Escherichia coli K-12 . J . Bacteriol . 184:1314-1323 .
36. Svetlichny, V . A., T . G . Sokolova, M . Gerhardt, M . Ringpfeil, N . A . Kostrikina, and G . A . Zavarzin. 1991 . Carboxydothermus hydrogenoformans gen . nov., sp . nov., a CO-utilizing thermophilic anaerobic bacterium from hydrothermal environments of Kunashir island . Syst . Appl . Microbiol . 14:254-260.
37. Svetlitchnyi, V., C . Peschel, G . Acker, and O . Meyer. 2001 . Two membrane-associated NiFeS-Carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans. J . Bacteriol . 183:5134-5144 .
38. Thorsteinsson, M . V., R . L . Kerby, and G . P . Roberts. 2000 . Altering the specificity of CooA, the carbon monoxide-sensing transcriptional activator: characterization of CooA variants that bind cyanide in the Fe^II form with high affinity . Biochemistry 39:8284-8290.
39. Thorsteinsson, M . V., R . L . Kerby, M . Conrad, H . Youn, C . R . Staples, W . N . Lanzilotta, T . J . Poulos, J . Serate, and G . P . Roberts. 2000 . Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator . J . Biol . Chem . 275:39332-39338 .
40. Uchida, T., H . Ishikawa, K . Ishimori, I . Morishima, H . Nakajima, S . Aono, Y . Mizutani, and T . Kitagawa. 2000 . Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy . Biochemistry 39:12747-12752.
41. Voordouw, G. 2002 . Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough . J . Bacteriol . 184:5903-5911 .
42. Waterman, M . R. 1978 . Spectral characterization of human hemoglobin and its derivatives . Methods Enzymol . 52:456-463.
43. Yamamoto, K., H . Ishikawa, S . Takahashi, K . Ishimori, I . Morishima, H . Nakajima, and S . Aono. 2001 . Binding of CO at the Pro² side is crucial for the activation of CO-sensing transcriptional activator CooA . ¹H NMR spectroscopic studies . J . Biol . Chem . 276:11473-11476 .
44. Youn, H., R . L . Kerby, M . V . Thorsteinsson, R . W . Clark, J . N . Burstyn, and G . P . Roberts. 2002 . Analysis of the L116K variant of CooA, the heme-containing CO sensor, suggests the presence of an unusual heme ligand resulting in novel activity . J . Biol . Chem . 277:33616-33623 .
45. Youn, H., R . L . Kerby, M . V . Thorsteinsson, M . Conrad, C . R . Staples, J . Serate, J . Beack, and G . P . Roberts. 2001 . The heme pocket afforded by Gly¹¹⁷ is crucial for proper heme ligation and activity of CooA . J . Biol . Chem . 276:41603-41610 .
46. Youn, H., R . L . Kerby, and G . P . Roberts. 2003 . The role of the hydrophobic distal heme pocket of CooA in ligand sensing and response . J . Biol . Chem . 278:2333-2340 .

Free Online Full-text Article

What Is Molecular Microbiology?, What Is Bioassay?, What Is MIC?, What Is Dna?, What Is Genome?, i, Microorganism, o, Microbe, e, Microbes, i, Bacterium, r, Microbiology, c, Microbial, e, Streptococci, r, Proteus, i, Cell suspensions, a, Microbial, n, Fermentations, s, Streptococci, e, Bacteriological, c, Meningococcus, o, Listeriosis, n, Microorganisms, r, Edwardsiella, e, Bacillus, n, Bacteria, s, Staphylococcus aureus, e, S. cerevisiae, c, Antibiotics, r, Candida albicans, r, Haemophilus, a, Microflora, a, Yeasts




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005