Christopher R. Staples

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

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 Pro2 of one subunit (Met1 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 resulting CooA variants. While Pro2appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro2 is replaced by Met1-Tyr2, for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe5 and Asn6 appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His77 ligand on the opposite side of the heme.

Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase in Azotobacter vinelandii*

The vnf-encoded nitrogenase fromAzotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in thevnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7–8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from anifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from thenifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

Carbon monoxide dehydrogenase from Rhodospirillum rubrum produces formate

Abstract. Carbon monoxide dehydrogenase (CODH) from Rhodospirillumrubrum reversibly catalyzes the oxidation of CO to CO2 at the active site C-cluster. In this article, the reduction of CO2 to formate is reported as a slow side reaction catalyzed by both Ni-containing CODH and Ni-deficient CODH. Recently, the structures of R. rubrum CODH and its active site NiFeS cluster (the C-cluster) have been solved. The data in this manuscript describe the formate-producing capability of CODH with or without Ni in the active site.