期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：13
页码：1
DOI：10.1073/pnas.2100921118
出版社：The National Academy of Sciences of the United States of America
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of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function.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 K M H 2 values, do not produce H 2 at pH 6, and display the same onset overpotential for H 2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O 2 tolerance (apparent K M H 2 and V max [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 − , H 2 , O 2 ) 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 H 2 oxidation by this enzyme.
