Peer Evaluation Meeting DOE Hydrogen and Fuel Cells Program Hydrogen Production / Basic Energy

P. furiosus obtains carbon and energy for growth by fermenting carbohydrates and producing H2 and by reducing elemental sulfur (S°) to H2S. It has a respiratory metabolism in which it couples H2 production by a ferredoxin-dependent, membrane-bound hydrogenase (MBH) to ion translocation and formation of a membrane potential that P. furiosus utilizes to synthesize ATP. P. furiosus also contains a cytoplasmic hydrogenase (SHI) that has the rare property of evolving H2 from NADPH, a reaction of utility in H2 production systems. Addition of S° to P. furiosus prevents the synthesis of MBH and SHI, and induces the synthesis of a highly homologous membrane complex which we term MBX. MBX is proposed to oxidize ferredoxin, reduce S° and conserve energy by an as yet unknown mechanism. The specific aims of this research are: 1) to characterize the novel energy-conserving complex MBH, 2) to characterize the novel energy-conserving complex MBX, and 3) to structurally characterize native SHI and minimal forms of SHI and MBH. We are taking advantage of our recent development of a genetic system in P. furiosus to generate strains containing deletions of key genes, affinity-tagged enzymes and/or over-expressed forms of the enzymes of interest. We are also heterologously expressing in P. furiosus H2-evolving membrane complexes from related archaea that are analogous to MBH. The results of this research will provide a fundamental understanding of energy conservation in P. furiosus that involve the metabolism of H2 gas.