The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

Resonance Raman spectra of the CO-responsive transcription factor CooA from Rhodospirillum rubrum provides evidence on the nature of heme ligation and its CO activation mechanism. The Fe(III) form gives standard low-spin heme spectrum, while the Fe(II) form is low spin for wild-type (WT) CooA and mixed spin for a CooA variant, H77Y, with an His77Tyr substitution. The Fe(II) porphyrin skeletal mode nu11 is at a value (1541 cm-1) indicative of a neutral donor ligand for the H77Y variant but is at an unusually depressed frequency (1529 cm-1) for the WT protein, indicating a strongly donating ligand. This ligand is proposed to be His77 imidazolate, formed by proton transfer to a nearby acceptor. The WT CO adduct has FeCO and CO stretching frequencies that indicate a neutral trans ligand and negative polarity in the CO binding pocket, while the CO adduct of His77Tyr has both 6- and 5-coordinate signals and a nonpolar CO environment. Photolysis of the WT CO adduct by the Raman laser produced a low-spin product at steady state, indicating fast recombination of the displaced ligand. These data suggest a novel YH- - -His- charge relay mechanism for CooA activation by CO. In this mechanism, His77 is reprotonated upon ligand displacement by CO; CO displaces either His77 or the trans ligand, X. The resulting charge on Y- may induce the protein conformation change required for site-selective DNA binding.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro 2 of one subunit (Met 1 is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the re- The sensing of dissolved gas molecules by proteins in biology has recently attracted considerable biochemical interest. The role of nitric oxide in a variety of important biochemical processes (1, 2), and its receptor, soluble guanylyl cyclase (sGC) 1 have been well documented in eukaryotic systems (3, 4). FixL, which modulates the expression of genes responsible for nitrogen fixation in rhizobia, is an example of an oxygen sensor (5, 6). An oxygen sensor in Escherichia coli, termed DOS ("direct oxygen sensor"), has been reported although its physiological role remains undefined (7). Finally, for carbon monoxide (CO), CooA, the CO-oxidation activator protein, modulates the expression of genes required for the utilization of CO as a sole energy source in the photosynthetic bacterium Rhodospirillum rubrum (8). All of the above mentioned proteins have in common a heme prosthetic group to which their respective gas molecules bind. The binding event is then followed by a conformational change in the protein that effects activity.Numerous studies have clearly demonstrated the physiological importance of CO in a wide variety of processes (9 -11), and although sGC has been implicated in sensing CO (12-14), direct evidence of a CO-receptor in eukaryotic signal transduction systems is lacking. CooA senses CO through a heme moiety and represents the current model system for biological CO-sensing (19,20). Finally, the ligand that is displaced upon binding CO remains speculative.Recently, the three-dimensional structure of Fe II CooA has been solved by x-ray diffraction techniques (21). This report showed that the general folding topology of CooA was indeed similar to that of CRP (22). In addition to the verification of His 77 as one of the heme-axial ligands in Fe II CooA, inspection of the structure identified the other axial ligand as an Nterminal proline residue (Pro 2 ; Met 1 is removed by processing) from the other subunit of the dimer. This structural environment represents an unprecedented axial ligation arrangement for a heme protein.In a previous study (18), we altered His 77 and found that the UV-visual spectra of these variants was normal in the Fe ʈ To whom correspondence should be addressed. Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts@bact.wisc.edu.1 The abbreviations used are: sGC, soluble guanylyl cyclase; CO, carbon monoxide; CRP, cAMP receptor protein; FixL, oxygen sensor of Rhizobium meliloti; Mb, myoglobin; P-4...

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n ‫؍‬ 1.4, P 50 ‫؍‬ 2.