Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module.
A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M (r) = 52 kDa) and a small (M (r) = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe-S clusters, three [2Fe-2S] clusters and five [4Fe-4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe-S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H(2)-dependent reduction of NAD(+). Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni-Fe active site and a [4Fe-4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni(a)-C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.
Long
Minnan
M
School of Life Sciences, Bio-energy Center, Xiamen University, Xiamen, 361005, People's Republic of China. longmn@xmu.edu.cn
Liu
Jingjing
J
Chen
Zhifeng
Z
Bleijlevens
Boris
B
Roseboom
Winfried
W
Albracht
Simon P J
SP
D000595:Amino Acid Sequence; D002841:Chromatiaceae / Q000201:enzymology* / Q000254:growth & development; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared; D013997:Time Factors
D004247:DNA; C043296:nickel-iron hydrogenase; D006864:Hydrogenase