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Journal of Bacteriology, April 2000, p. 1864-1871, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Kesen Ma,1,dagger Robert Weiss,2 and Michael W. W. Adams1,*

Department of Biochemistry and Molecular Biology, Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602,1 and Department of Genetics, University of Utah, Salt Lake City, Utah 841122

Received 26 August 1999/Accepted 13 January 2000

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alpha beta gamma delta ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H2 production and the reduction of elemental sulfur to H2S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an Mr of 320,000 ± 20,000 and contains four different subunits with Mrs of 52,000 (alpha ), 39,000 (beta ), 30,000 (gamma ), and 24,000 (delta ). 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 H2 production with Km values near 70 µM and kcat/Km values near 350 min-1 mM-1. In contrast to hydrogenase I, hydrogenase II catalyzed the H2-dependent reduction of NAD (Km, 128 µM; kcat/Km, 770 min-1 mM-1). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H2 and NADPH served as electron donors for the reduction of elemental sulfur (S0) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H2 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) and one [2Fe-2S] cluster (in the gamma  subunit), as well as two putative nucleotide-binding sites in the gamma  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 S0 concentrations since it has a higher affinity than hydrogenase I for both S0 and polysulfide.

* Corresponding author. Mailing address: Department of Biochemistry, Life Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.

dagger Present address: Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.

Journal of Bacteriology, April 2000, p. 1864-1871, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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