CO-dependent H 2 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 4S 4 clusters. The observation of 2 Fe 4S 4 clusters for CooF contradicts the prediction of 4 Fe 4S 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 ∼ 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.