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

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