title: CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex.
abstract: Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity.
authors.affiliation: Department of Plant and Microbial Biology, University of California-Berkeley, 111 Koshland Hall, Berkeley, CA 94720-3102, USA.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Singer
authors.forename: Marissa B
authors.initials: MB
authors.lastname: Hirst
authors.forename: Paul W
authors.initials: PW
authors.lastname: Ludden
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D006859:Hydrogen / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes; D021122:Protein Subunits; D018085:Regulon; D012247:Rhodospirillum rubrum / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D046912:Multiprotein Complexes; D021122:Protein Subunits; D002248:Carbon Monoxide; D006859:Hydrogen; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase