Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-uti lizing, heterotetrameric (alpha beta gamma delta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H-2 production and the reduction of ele mental sulfur to H2S, Herein is described the purification of a second enzy me of this type, hydrogenase II, from the same organism. Hydrogenase II has an M-r of 320,000 +/- 20,000 and contains four different subunits with M(r )s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). Th e heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/ mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH a nd NADH were equally efficient as electron donors for H-2 production with K -m values near 70 mu M and k(cat)/K-m values near 350 min(-1) mM(-1), In co ntrast to hydrogenase I, hydrogenase II: catalyzed the H-2-dependent reduct ion of NAD (K-m, 128 mu M; k(cat)/K-m, 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H-2 and NADPH served as electron donors for the reduction of elemental sulf ur (S-0) and polysulfide by hydrogenase I and hydrogenase II, and both enzy mes preferentially reduce polysulfide to sulfide rather than protons to H-2 , using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [ 4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) a nd one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nue leotide-binding sites in the gamma subunit which are thought to bind FAD an d NAD(P)(H), The amino acid sequences of the four subunits of hydrogenase I I showed between 55 and 63% similarity to those of hydrogenase I. The two e nzymes are present in the cytoplasm at approximately the same concentration . Hydrogenase II: may become physiologically relevant at low S-0 concentrat ions since it has a higher affinity than hydrogenase I for both S-0 and pol ysulfide.