Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module |
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Authors: | Minnan Long Jingjing Liu Zhifeng Chen Boris Bleijlevens Winfried Roseboom Simon P J Albracht |
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Institution: | (1) School of Life Sciences, Bio-energy Center, Xiamen University, Xiamen, 361005, People’s Republic of China;(2) Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands |
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Abstract: | 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 H2-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 Nia–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of
NiFe] hydrogenase. |
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Keywords: | Soluble [NiFe] hydrogenase Allochromatium vinosum Purification Sequence NADH:ubiquinone oxidoreductase |
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