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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.
Afiliación
  • Evans RM; Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom.
  • Krahn N; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
  • Murphy BJ; Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom.
  • Lee H; Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom.
  • Armstrong FA; Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom; fraser.armstrong@chem.ox.ac.uk dieter.soll@yale.edu.
  • Söll D; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511; fraser.armstrong@chem.ox.ac.uk dieter.soll@yale.edu.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Article en En | MEDLINE | ID: mdl-33753519
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of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function.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.
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Oxígeno / Selenocisteína / Cisteína / Proteínas de Escherichia coli / Hidrogenasas Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2021 Tipo del documento: Article País de afiliación: Reino Unido

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Oxígeno / Selenocisteína / Cisteína / Proteínas de Escherichia coli / Hidrogenasas Idioma: En Revista: Proc Natl Acad Sci U S A Año: 2021 Tipo del documento: Article País de afiliación: Reino Unido