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Enzymatic and spectroscopic properties of a thermostable [NiFe]-hydrogenase performing H-2-driven NAD(+)-reduction in the presence of O-2
Zitatschlüssel ISI:000418314600002
Autor Preissler, Janina and Wahlefeld, Stefan and Lorent, Christian and Teutloff, Christian and Horch, Marius and Lauterbach, Lars and Cramer, Stephen P. and Zebger, Ingo and Lenz, Oliver
Seiten 8-18
Jahr 2018
ISSN 0005-2728
DOI 10.1016/j.bbabio.2017.09.006
Journal BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Jahrgang 1859
Nummer 1
Monat JAN
Zusammenfassung Biocatalysts that mediate the H-2-dependent reduction of NAD(+) to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD(+)-reducing [NiFe]-hydrogenase that sustains catalytic activity at high temperatures and in the presence of O-2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD(+)-reducing [NiFe]-hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1(T) (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well studied, H-2-oxidizing betaproteobacterium Raistonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H-2-mediated NAD(+) reduction activity was observed at 80 degrees C and pH 6.5, and catalytic activity was found to be sustained at low O-2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]-hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD(+)-reducing [NiFe]-hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H-2-driven cofactor recycling under oxic conditions at elevated temperatures.
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