Impact of the Iron鈥揝ulfur Cluster Proximal to the Active Site on the Catalytic Function of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase

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• 作者：Katja Karstens ; Stefan Wahlefeld ; Marius Horch ; Miriam Grunzel ; Lars Lauterbach ; Friedhelm Lendzian ; Ingo Zebger ; Oliver Lenz
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：389-403
• 全文大小：622K
• ISSN：1520-4995

文摘

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 O₂ 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鈥搒ulfur 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 O₂-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O₂ sensitity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O₂-tolerant SH representatives did not greatly affect the H₂-oxidizing activity in the presence of O₂. 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鈥搒ulfur 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 H₂/D⁺ exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.