Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

Laun, Konstantin; Baranova, Iuliia; Duan, Jifu; Kertess, Leonie; Wittkamp, Florian; Apfel, Ulf‐Peter; Happe, Thomas; Senger, Moritz; Stripp, Sven T.

doi:10.1039/d1dt00110h

Cited by 18 publications

(30 citation statements)

References 62 publications

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“…67 In a recent study, we addressed the influence of pH on the accumulation of reduced H-cluster states under turnover conditions. 55 We noted a clear trend for Hred to dominate at pH < 7 and Hred′ to dominate at pH > 7, confirming former observations. 54 This can explain the bell-shaped activity distribution as a function of pH: the preference for Hred under proton-rich conditions might indicate that the H-cluster forms an unready, H 2 -inhibited geometry upon PCET to the Fe−Fe cofactor, e.g., controlled by the low pK a of the glutamic acid residues in the PT pathway.…”

Section: Understanding [Fefe]-hydrogenase Catalysissupporting

confidence: 90%

Proton Transfer Mechanisms in Bimetallic Hydrogenases

2020

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Section: Understanding [Fefe]-hydrogenase Catalysissupporting

confidence: 90%

Proton Transfer Mechanisms in Bimetallic Hydrogenases

2020

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“…Both 1e – -reduced states H red′ and H red are formed upon proton-coupled electron transfer and can be distinguished in FTIR spectroelectrochemistry due to their different p K a values Figure compares two difference spectra recorded in potential-jump experiments at (a) pH 9 and (b) 5 . Under alkaline conditions and reducing potentials (−650 mV vs SHE), H ox converts exclusively into H red′ .…”

Section: Complementary In Situ Approachesmentioning

confidence: 99%

“…128 Figure 15 compares two difference spectra recorded in potential-jump experiments at (a) pH 9 and (b) 5. 129 Under alkaline conditions and reducing potentials (−650 mV vs SHE), H ox converts exclusively into H red′ . On the opposite, H red converts exclusively into H sred under acidic conditions and strongly reducing potentials (−750 mV vs SHE).…”

Section: Complementary In Situ Approachesmentioning

confidence: 99%

In Situ Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

Stripp

2021

ACS Catal.

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Earth-abundant transition metals, such as iron, nickel, copper, molybdenum, and vanadium, have been identified as essential constituents of the cellular gas metabolism in all kingdoms of life. Associated with biological macromolecules, gas-processing metalloenzymes (GPMs) are formed that catalyze a variety of redox reactions. This includes the reduction of O2 to water by cytochrome c oxidase (“complex IV”), the reduction of N2 to NH3 by nitrogenase, as well as the reversible reduction of protons to H2 by hydrogenase. GPMs perform at ambient temperature and pressure, in the presence of water, and often extremely low educt concentrations, thus serving as natural examples for efficient catalysis. Facilitating the design of biomimetic catalysts, biophysicist thrive to understand the reaction principles of GPMs making use of various techniques. In this Perspective, I will introduce Fourier-transform infrared spectroscopy in attenuated total reflection configuration (ATR FTIR) for the analysis of GPMs like cytochrome c oxidase, nitrogenase, and hydrogenase. Infrared spectroscopy provides information about the geometry and redox state of the catalytic cofactors, the protonation state of amino acid residues, the hydrogen-bonding network, and protein structural changes. I developed an approach to probe and trigger the reaction of GPMs by gas exchange and deuteration experiments exploring the reactivity of these enzymes with their natural reactants. This allows recording sensitive steady-state ATR FTIR (difference) spectra with seconds time resolution. Finally yet importantly, infrared spectroscopy is an electronically noninvasive technique that allows investigating protein samples under biologically relevant conditions, that is, at ambient temperature, ambient pressure, and in the presence of liquid water.

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“…S3). [16] While [FeFe]-hydrogenases prepared at pH 4 have been shown to adopt HoxH as main oxidized resting state in the presence of NaDT [20][21][22] , we now found that HoxH is not observed at pH 4 when CrHydA1 was mixed with TCEP, DTT, ascorbic acid, or no reductant (Fig. S2A).…”

Section: Resultsmentioning

confidence: 60%

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions

Senger

Duan

Pavliuk

et al. 2022

Preprint

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The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the production of hydrogen gas (H2). The H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from the oxidized resting state (Hox) by a small but distinct shift to higher frequencies. This ‘blue shift’ was explained by a protonation at the [4Fe-4S] sub-complex of the H-cluster. The novel species, denoted HoxH, was preferentially accumulated at low pH and in the presence of the exogenous reductant sodium dithionite (NaDT). When HoxH was reacted with H2, the hydride state (Hhyd) was formed, a key intermediate of [FeFe]-hydrogenase turnover. A recent publication revisited our protocol for the accumulation of HoxH in wild-type [FeFe]-hydrogenase, concluding that inhibition by NaDT decay products rather than cofactor protonation causes the spectroscopic ‘blue shift’. Here, we demonstrate that HoxH formation does not require the presence of NaDT (or its decay products), but accumulates also with the milder reductants tris(2-carboxyethyl)phosphine, dithiothreitol, or ascorbic acid, in particular at low pH. Our data consistently suggest that HoxH is accumulated when deprotonation of the H-cluster is impaired, thereby preventing the regain of the oxidized resting state Hox in the catalytic cycle.

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Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

Abstract: Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H2 oxidation activity, [FeFe]-hydrogenases are excellent H2 evolution catalysts as...

Cited by 18 publications

References 62 publications

Proton Transfer Mechanisms in Bimetallic Hydrogenases

Proton Transfer Mechanisms in Bimetallic Hydrogenases

In Situ Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions

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