Characterization of Hydrogenase II from the Hyperthermophilic Archaeon Pyrococcus furiosus and Assessment of Its Role in Sulfur Reduction

Abstract

ABSTRACT The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H 2 production and the reduction of elemental sulfur to H 2 S. Herein is described the purification of a second enzyme 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 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The 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 and NADH were equally efficient as electron donors for H 2 production with K m values near 70 μM and k cat / K m values near 350 min −1 mM −1 . In contrast to hydrogenase I, hydrogenase II catalyzed the H 2 -dependent reduction of NAD ( K m , 128 μM; 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 sulfur (S 0 ) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes 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 β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S 0 concentrations since it has a higher affinity than hydrogenase I for both S 0 and polysulfide.