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Published January 10, 2017 | Supplemental Material
Journal Article Open

Importance of the Active Site "Canopy" Residues in an O_2-Tolerant [NiFe]-Hydrogenase

Abstract

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O_2 interact, substrate H_2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H_2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H_2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H_2/D_2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H_2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H_2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O_2 tolerance by stabilizing the oxidized resting Ni^(III)–OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.

Additional Information

© 2016 American Chemical Society. Received: August 28, 2016; Revised: November 21, 2016; Publication Date (Web): December 21, 2016. We thank Diamond Light Source for beam time (proposal mx12346) and the staff of beamline I04-1 for their assistance. We thank Elena Nomerotskaia for invaluable technical assistance throughout this work. This research was supported by the UK Biological and Biotechnology Sciences Research Council (Grants BB/I022309-1 and BB/L009722/1 to F.A.A.). A studentship for E.J.B. was supported by grants from Global Innovation Initiative and the UK Engineering and Physical Sciences Research Council. The Islamic Development Bank provided a studentship for S.T.A.I. under the Merit Scholarship Programme for High Technology. F.A.A. is a Royal Society-Wolfson Research Merit Award holder. We are also that the Margaret Leighton SURF Fellowship (California Institute of Technology) provided a visiting studentship to G.M.R. The authors declare no competing financial interest.

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Created:
August 19, 2023
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October 24, 2023