Purification and Molecular Characterization of an [FeFe]-Hydrogenase from Thermotoga hypogea

Title:Purification and Molecular Characterization of an [FeFe]-Hydrogenase from Thermotoga hypogea

Volume: 4 Issue: 2

Author(s): Xianqin Yang, Liangliang Hao, Hongbin Zhu and Kesen Ma

Affiliation:

Keywords: [FeFe]-hydrogenase, ferredoxin, iron-sulfur clusters, ferredoxin, hydrogen production, hyperthermophiles, Thermotoga hypogea.

Abstract: Thermotoga hypogea is a hyperthermophilic bacterium growing on carbohydrates and peptides, producing acetate, CO₂ and H₂ as major end products. Hydrogenase activity was detected in the cell-free extract of T. hypogea and a hydrogenase was purified to homogeneity. The purified enzyme was homotetrameric with a subunit of 65 kDa, and contained 16 atoms of Fe and 11.6 atoms of acid labile sulfur per mole subunit. The gene encoding the hydrogenase was sequenced, and four iron-sulfur clusters including the H-cluster were found to be present in the gene sequence, indicating it is an [FeFe]-hydrogenase. The enzyme was oxygen sensitive with a t_1/2 of 3 min when exposed to air. It was thermostable with t_1/2 values of approximately 40 and 15 min when incubated at 70°C and 85°C, respectively. The optimal temperature for the hydrogenase activity was about 85 °C. Both benzyl viologen (BV) and methyl viologen (MV) could serve as electron acceptors, and the apparent V_max values were determined to be 1,142 and 607 μmol H2 oxidized min^{-1 mg-1, respectively. For H₂ uptake activity, the apparent K_m values for MV and BV were 0.17 and 0.24 mM, respectively. For H2 evolution activity, the apparent K_m value for MV and apparent V_max value were determined to be 1.1 mM and 192 μmol min-1 mg-1, respectively. Ferredoxin purified from T. hypogea served as electron carrier. The enzyme exhibited pH optima of 10.0 and 8.0 for the H2 uptake and evolution activities, respectively. The physiological function of the enzyme is proposed to catalyze the H₂ evolution.}

Cite this article as:

Yang Xianqin, Hao Liangliang, Zhu Hongbin and Ma Kesen, Purification and Molecular Characterization of an [FeFe]-Hydrogenase from Thermotoga hypogea, Current Biotechnology 2015; 4 (2) . https://dx.doi.org/10.2174/2211550104666150414185950