Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Abstract

ABSTRACT We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 ( T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames ( hyhBGSL Tk ), where the hyhS Tk and hyhL Tk gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase ( hybD Tk ) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL Tk were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (β, γ, δ, and α), corresponding to the products of hyhBGSL Tk , respectively. Each αβγδ unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95°C for H 2 uptake and 90°C for H 2 production with methyl viologen as the electron carrier. We found that NADP + and NADPH promoted high levels of uptake and evolution of H 2 , respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.