CO-dependent H2 evolution by Rhodospirillum rubrum:: Role of CODH:CooF complex

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 Fe4S4 clusters. The observation of 2 Fe4S4 clusters for CooF contradicts the prediction of 4 Fe4S4 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 similar to 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. (c) 2006 Elsevier B.V. All rights reserved.