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Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase.

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posted on 2019-06-18, 08:14 authored by RM Evans, PA Ash, SE Beaton, EJ Brooke, KA Vincent, SB Carr, FA Armstrong
Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Nia-R" and "Nia-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.

Funding

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 (EPSRC). F.A.A. is a Royal Society Wolfson Research Merit Award holder. The work of K.A.V. and P.A.A. was supported by EPSRC grant EP/N013514/1. We are grateful to Amelia Brasnett for carrying out initial PFIRE experiments on E28Q Hyd-1 during a summer internship. We thank Diamond Light Source for beam-time (proposal mx12346) and staff at beamlines I04-1, I03, and I24 for assistance during X-ray data collection.

History

Citation

Journal of the American Chemical Society, 2018, 140 (32), pp. 10208-10220

Author affiliation

/Organisation/COLLEGE OF SCIENCE AND ENGINEERING/Department of Chemistry

Version

  • VoR (Version of Record)

Published in

Journal of the American Chemical Society

Publisher

American Chemical Society

eissn

1520-5126

Copyright date

2018

Available date

2019-06-18

Publisher version

https://pubs.acs.org/doi/10.1021/jacs.8b04798

Notes

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b04798. Denaturing electrophoresis; plasmid, strains and primer details; PFIRE film preparation; initial E28Q PFE data; additional supporting PFE of E28Q on modified electrodes; PFE data for E28D variant; steady-state solution assay data; X-ray diffraction collection and crystal structure statistics; additional structural figures; PFIRE cyclic voltammograms, chronoamperometry and IR spectra of E28Q (PDF) pdf ja8b04798_si_001.pdf (1.21 MB)

Language

en

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