Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

Evans, Rhiannon M.; Krahn, Natalie; Murphy, Bonnie J.; Lee, Harrison; Armstrong, Fraser A.; Söll, Dieter

doi:10.1073/pnas.2100921118

Title: Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

Journal Article · Mon Mar 22 00:00:00 EDT 2021 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2100921118· OSTI ID:1772179

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Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom,
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511,
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511,, Department of Chemistry, Yale University, New Haven, CT 06520

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of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function.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_MH₂ 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 K_MH₂ 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^-, 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.

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Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); UK Biological and Biotechnology Sciences Research Council

Grant/Contract Number:: FG0298ER2031; FG02-98ER2031; OD01803; BB/I022309-1; BB/L009722/1; R35GM122560

OSTI ID:: 1772179

Alternate ID(s):: OSTI ID: 1817005

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 118 Journal Issue: 13; ISSN 0027-8424

Publisher:: Proceedings of the National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
selenocysteine
hydrogenase
hydrogen activation
oxygen tolerance

Title: Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

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