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Impact of the Iron-Sulfur Cluster Proximal to the Active Site on the Catalytic Function of an O-2-Tolerant NAD(+)-Reducing [NiFe]-Hydrogenase
Zitatschlüssel ISI:000348333300028
Autor Karstens, Katja and Wahlefeld, Stefan and Horch, Marius and Grunzel, Miriam and Lauterbach, Lars and Lendzian, Friedhelm and Zebger, Ingo and Lenz, Oliver
Seiten 389-403
Jahr 2015
ISSN 0006-2960
DOI 10.1021/bi501347u
Adresse 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
Journal BIOCHEMISTRY
Jahrgang 54
Nummer 2
Monat JAN 20
Verlag AMER CHEMICAL SOC
Zusammenfassung The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O-2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O-2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O-2 sensitity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O-2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O-2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H-2/D+ exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.
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