A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

Abou‐Hamdan, Abbas; Ceccaldi, P F; Lebrette, Hugo; Gutiérrez‐Sanz, Óscar; Richaud, Pierre; Cournac, Laurent; Guigliarelli, Bruno; Lacey, Antonio L. De; Léger, Christophe; Volbeda, Anne; Burlat, Bénédicte; Dementin, Sébastien

doi:10.1074/jbc.m114.630491

Cited by 18 publications

(14 citation statements)

References 45 publications

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“…Rotation of the equivalent aspartate residue about the Cα−Cβ bond has also been observed in a T18V variant of the O 2 -sensitive [NiFe]hydrogenase from D. fructosovorans but with no addition of OH − near residue 28. 68 The affinity of OH − for occupying the position near to the amide of Q28 thus depends on two factors: (1) the proximal cluster being in the superoxidized form or, (2) providing a high pH, even when the proximal cluster is reduced.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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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 O-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 H oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H 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 "Ni-R" and "Ni-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H, but facilitates H exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Ni-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H at high potential because Ni-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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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“…As shown by Figure 9 (A), over the past 10 years substantial progress has been made in the determination of NiFe hydrogenase X-ray structures. Enzymes from both aerobic ( Hydrogenovibrio marinus [ 58 ] and R. eutropha [ 59 , 60 ]), anaerobic ( Desulfovibrio gigas [ 48 , 51 ], Desulfovibrio fructosovorans [ 53 , 54 , 61 ], Desulfovibrio vulgaris [ 52 , 62 – 64 ], Desulfomicrobium baculatum [ 65 , 66 ] and A. vinosum [ 67 ]) and facultative anaerobic organisms ( E. coli [ 68 , 69 ] and S. enterica [ 70 ]) have been crystallized ( Figure 9 B) [ 71 ], resulting in the elucidation of the heterodimer structures of several O 2 sensitive and O 2 tolerant NiFe hydrogenases. The overall structure of all these enzymes is remarkably similar ( Figure 10 ).…”

Section: The Essential Role Of the Electron-transfer Relaymentioning

confidence: 99%

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

Flanagan

Parkin

2016

Biochemical Society Transactions

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Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH.

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“… 35 More transient states have also been probed in time-resolved infrared spectroscopic techniques in which transitions between states of a hydrogenase have been triggered by the release of caged electrons or by photolysis of light-sensitive states of the enzyme. 41 , 42 , 60 Attention has also focused on the role of conserved amino acids in the region around the active site in controlling proton transfer, 12 , 54 , 61 − 63 and the correlation between the redox state of the proximal cluster and that of the active site. 36 , 60 These studies have been aided by the availability of site-directed variants of [NiFe] hydrogenases with particular amino acid exchanges in the region of the active site and the proximal iron sulfur cluster.…”

Section: Introductionmentioning

confidence: 99%

“… 36 , 60 These studies have been aided by the availability of site-directed variants of [NiFe] hydrogenases with particular amino acid exchanges in the region of the active site and the proximal iron sulfur cluster. 12 , 54 , 61 , 63 However, one limitation to recent studies of [NiFe] hydrogenases has been that each vibrational spectroscopy approach has only been applied to one or two different hydrogenases, and it remains to be seen whether the findings from each approach represent aspects of a common [NiFe] hydrogenase mechanism or whether certain details are specific to individual hydrogenases.…”

Section: Introductionmentioning

confidence: 99%

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

2017

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Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases.

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A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

Cited by 18 publications

References 45 publications

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

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

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

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A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

Cited by 18 publications

References 45 publications

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

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

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

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

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