Enzymes of hydrogen metabolism in Pyrococcus furiosus

Abstract

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni±Fe hydrogenase. Ten ORFs (mbhA±I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni±Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe±4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe±4S] protein. With P. furiosus soluble [4Fe±4S] ferredoxin as the electron donor the membranes produce H 2 , and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H 2 evolution to H 2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E m < 20.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni±Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H 2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H 2 ) connected by devices for transduction, transfer, recovery and safety-valving of energy.

Recombination between elongation factor 1α genes from distantly related archaeal lineages

“…The first recombination case we described involves Methanopyrus and the Thermococcales, organisms that both were originally isolated from deep-sea hyperthermal vents and grow at nearly 100°C. It is even possible that some members of these groups exist in a symbiotic relationship, because Pyrococcus produces hydrogen as an end product of respiration, whereas Methanopyrus utilizes hydrogen for energy production (27). A similar argument can be made for the second example we describe because the methanogenic archaeal lineages involved are both strictly anaerobic and grow at 40-70°C.…”

Insights on the Evolution of Metabolic Networks of Unicellular Translationally Biased Organisms from Transcriptomic Data and Sequence Analysis

“…Ferredoxin appears to be the major metabolic electron carrier in pyrococci (Cohen et al 2003). After reduction during peptide or sugar degradation, it is mainly reoxidized by a membrane-bound hydrogenase (Silva et al 2000), potentially generating membrane potential. In addition, ferredoxin has been suggested to be reoxidized by ferredoxin-NADP oxidoreductase (Silva et al 2000;Schut et al 2001).…”

“…After reduction during peptide or sugar degradation, it is mainly reoxidized by a membrane-bound hydrogenase (Silva et al 2000), potentially generating membrane potential. In addition, ferredoxin has been suggested to be reoxidized by ferredoxin-NADP oxidoreductase (Silva et al 2000;Schut et al 2001). Moreover, NADPH can also be oxidized via the conversion of pyruvate to alanine, via glutamate dehydrogenase (CAI(PAB0391)=0.74) and alanine aminotransferase (CAI(PAB1810)=0.55) (Ward et al 2000).…”

Molecular Biology of Microbial Hydrogenases

“…The majority of H 2 ases assigned to group 4 have been found in Archaea (Figure 1), including Methanosarcina barkeri (Künkel et al, 1998), Methanobacterium thermoautotrophicum strain Marburg (now called Methanothermobacter marburgensis) (Tersteegen and Hedderich, 1999) and Pyrococcus furiosus (Sapra et al, 2000;Silva et al, 2000). The membrane-bound [NiFe]-H 2 ase found in the acetate-grown methanogenic archaeon M. barkeri, catalyzes H 2 formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO 2 , by an energy-conserving mechanism (Künkel et al, 1998;Meuer et al, 1999Meuer et al, , 2002.…”

“…The methanogenic archaeon M. thermoautotrophicum, which is able to grow on CO 2 / H 2 as carbon and energy source contains, in addition to F 420 -reducing and F 420 -non-reducing H 2 ases, two gene groups designated "energy converting H 2 ase A" (eha) and "energy converting H 2 ase B" (ehb), which encode putative, multisubunit, membrane-bound H 2 ases homologous to E. coli H 2 ase-3 and R. rubrum CooHL H 2 ase (Tersteegen and Hedderich, 1999). The hyperthermophilic archaeon P. furiosus contains two cytoplasmic H 2 -evolving H 2 ases (I and II) (Pedroni et al, 1995;Ma et al, 2000), members of group 3, and a membrane-bound H 2 ase (MBH), member of group 4, encoded by a 14-gene operon (Schut et al, 2001) termed mbh (either mbh1-14 (Sapra et al, 2000) or mbhA-N (Silva et al, 2000)). Four gene products of this operon share similarities with subunits of complex I.…”