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

Rhiannon M Evans,Stephen B Carr,Kylie A Vincent,Emily J Brooke,Fraser A Armstrong,Philip A Ash,Stephen E Beaton

doi:10.1021/jacs.8b04798

Rhiannon M Evans, Stephen B Carr + Show 5 more

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

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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.

Highlights

The cycling connections of to hybdiorotegcehnno(Hlog2)y,byenmerigcryooarngdanihsmeaslthh.a1s−3strTonhge metalloenzymes known as hydrogenases offer a paradigm for fast, efficient and reversible[4] hydrogen electrocatalysis requiring only a minimal overpotential in either direction, and set a very high standard for designing catalysts based on abundant, nonplatinum metals.[5−7]Molecular H2 activation is the simplest of all proton-coupled electron-transfer reactions
Protein film infrared electrochemistry was carried out using a spectroelectrochemical flow cell as described previously,[33,34] and mass transport limitation was avoided by using a high solution flow rate (≥60 mL/min) through the Protein film infrared (IR) electrochemistry (PFIRE) cell via a peristaltic pump (Whatman, 120U/D1)
The second catalytic regime seen with E28Q, which results in a 3-fold increase in current at +0.6 V relative to that at +0.05 V, is due to enzymatic H2 oxidation: switching to 100% Ar causes the current to drop to background levels (Figure 2B) and returning to 100% H2 at ∼ +0.3 V immediately restores the current, which is retained upon subsequent scans

Summary

INTRODUCTION

The cycling connections of to hybdiorotegcehnno(Hlog2)y,byenmerigcryooarngdanihsmeaslthh.a1s−3strTonhge metalloenzymes known as hydrogenases offer a paradigm for fast, efficient and reversible[4] hydrogen electrocatalysis requiring only a minimal overpotential in either direction, and set a very high standard for designing catalysts based on abundant, nonplatinum metals.[5−7]. Hydrogenases operate in neutral water and use a heterolytic mechanism (H2 ⇌ H+ + H− ⇌ 2H+ + 2e−)[8] so it is highly significant that their active sites are deeply buried and almost completely sealed from solvent They are remarkable among all enzymes in that H2, the smallest of molecules, is solely formed from (or converted into) four quantum particles that must hop and tunnel through the protein. The Fe−S chain comprises three Fe−S clusters termed “proximal”, “medial” and “distal” according to their distance from the active site In contrast to this clearly marked pathway for electrons, our insight into long-range proton transfer owes much to the seminal work of Dementin and co-workers[24] who showed that a highly conserved glutamate[23] The results have important significance for understanding the mechanism of enzymatic H2 oxidation and how catalysis is linked to long-range proton transfer

EXPERIMENTAL SECTION

RESULTS

DISCUSSION AND CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES