气候变化领域集成服务门户(CCPortal): Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

CCPortal

DOI	10.1073/pnas.2100921118
	Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance
	Evans R.M.; Krahn N.; Murphy B.J.; Lee H.; Armstrong F.A.; Söll D.
发表日期	2021
ISSN	00278424
卷号	118 期号:13
英文摘要	In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent KMH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent KMH2 and Vmax [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H−, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Hydrogen activation; Hydrogenase; Oxygen tolerance; Selenocysteine
语种	英语
scopus关键词	cysteine; hydrogen; hydrogenase; nickel iron hydrogenase 1; oxygen; selenocysteine; unclassified drug; Article; catalysis; chemical binding; concentration (parameter); conformational transition; controlled study; electrochemistry; enzyme active site; enzyme activity; enzyme metabolism; Escherichia coli; mass spectrometry; nonhuman; priority journal; proton transport; steady state
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180061
作者单位	Inorganic Chemistry Laboratory, University of Oxford, Oxford, OX1 3QR, United Kingdom; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, United States; Department of Chemistry, Yale University, New Haven, CT 06520, United States
推荐引用方式 GB/T 7714	Evans R.M.,Krahn N.,Murphy B.J.,et al. Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance[J],2021,118(13).
APA	Evans R.M.,Krahn N.,Murphy B.J.,Lee H.,Armstrong F.A.,&Söll D..(2021).Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance.Proceedings of the National Academy of Sciences of the United States of America,118(13).
MLA	Evans R.M.,et al."Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance".Proceedings of the National Academy of Sciences of the United States of America 118.13(2021).