Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

摘要: Hydrogenases catalyze the reversible reduction of protons to hydrogen gas. They are found in a wide variety microorganisms and enable them use H2 as source reductant under either aerobic or anaerobic conditions. Alternatively, fermentative-type organisms utilize hydrogenase dispose without need terminal electron acceptors other than (1, 3). can be divided into two major types, depending on metals they contain (5). The so-called iron-only hydrogenases have high specific activities usually function evolve H2. Their catalytic site is comprised novel 6Fe cluster (26, 29). active nickel- iron-containing (NiFe-hydrogenases), hand, consists binuclear NiFe center (12, 36). NiFe-hydrogenases less their Fe-only counterparts, physiological role oxidize In H2-oxidizing bacteria, both cytoplasmic, NAD-reducing enzymes part conventional membrane-bound (MB) respiratory chains where O2 acceptor (4, 9). contrast, systems, poorly understood. For example, methanogen Methanosarcina barkeri contains an MB NiFe-hydrogenase multiprotein complex (18, 25), components which show sequence similarity NiFe-hydrogenase-containing present photosynthetic bacterium Rhodospirillum rubrum (13, 14). Both these systems thought involved energy conservation, but pathways transfer precise associated proteins unclear. addition, three four Escherichia coli also conservation (7, 8, 34). In this study, we focused metabolism by archaeon Pyrococcus furiosus, obligate organotroph that grows optimally near 100°C (11). This fermentative organism utilizes sugars via modified ADP-dependent Embden-Meyerhof pathway (16), while amino acids derived from peptides metabolized transaminases suite 2-keto acid oxidoreductases (2). conserved substrate-level phosphorylation. coenzyme A (CoA) derivatives generated converted organic directly pair enzymes, acetyl-CoA synthetases I II, simultaneously convert ADP phosphate ATP (23). end products fermentation acetate, H2, CO2; some produced when growth substrate. oxidation acid-derived glyceraldehyde-3-phosphate pyruvate glucose all carried out ferredoxin-dependent oxidoreductases. It has been proposed (22) reduced ferredoxin coupled NADP ferredoxin: oxidoreductase (FNOR [19]) NADPH then serves donor cytoplasmic H2-evolving (I II) (10, 21, 28). reason why such not understood (21). P. furiosus reduces elemental sulfur (S0) H2S. process decreases amount stimulatory effect growth, indicated increase cell density rate Moreover, during maltose, yield per gram substrate used 50% higher if S0 medium (35). suggests P. merely means disposing excess rather energy-conserving process. To date capable reducing H2S purified furiosus. These aforementioned FNOR, referred sulfide dehydrogenase (19), hydrogenases, otherwise known sulfhydrogenases (20, 21). However, located cytoplasm, it seems unlikely would conservation. In effort determine whether reductase system analogous S0-respiring mesophile Wolinella succinogenes (15, 30), sought obtain membrane fraction extracts lacked H2-dependent, activity sulfhydrogenases. Surprisingly, even after repeated washings with buffers containing salt concentrations, still contained (H2 evolution) activity. purification characterization integral described herein. enzyme type, functions does reduce S0, distinct well-characterized enzyme. appears large multienzyme complex, respiratory-linked, methanogens bacteria nonenergy-conserving formate lyase (hydrogenase 3) E. (34).