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Title: Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase.
Authors: Evans, RM
Ash, PA
Beaton, SE
Brooke, EJ
Vincent, KA
Carr, SB
Armstrong, FA
First Published: 2-Aug-2018
Publisher: American Chemical Society
Citation: Journal of the American Chemical Society, 2018, 140 (32), pp. 10208-10220
Abstract: 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.
DOI Link: 10.1021/jacs.8b04798
eISSN: 1520-5126
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © the authors, 2018. This is an open-access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Description: 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)
Appears in Collections:Published Articles, Dept. of Chemistry

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