Chalcogenide substitution in the [2Fe] cluster of [FeFe]-hydrogenases conserves high enzymatic activity

Kertess, Leonie; Wittkamp, Frank; Sommer, Constanze; Esselborn, Julian; Rüdiger, Olaf; Reijerse, Edward J.; Hofmann, Eckhard; Lubitz, Wolfgang; Winkler, Martin; Happe, Thomas; Apfel, Ulf‐Peter

doi:10.1039/c7dt03785f

Cited by 52 publications

(66 citation statements)

References 59 publications

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“…We subsequently took the next step and replaced the bridging thiolates of the [2Fe] H cluster by selenides. 43 In line with reports on chalcogenide replacements within synthetic mimics, we expected that this S/Se exchange will lead to enhanced catalytic activity, e.g. due to an increase of electron density at the diiron site and optimized electron transfer, accordingly.…”

Section: Artificial Maturationsupporting

confidence: 70%

[FeFe]-Hydrogenases: recent developments and future perspectives

et al. 2018

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[FeFe]-Hydrogenases are the most efficient enzymes for catalytic hydrogen turnover. Their H2 production efficiency is hitherto unrivalled. However, functional details of the catalytic machinery and possible modes of application are discussed controversially. The incorporation of synthetically modified cofactors and utilization of semi-artificial enzymes only recently allowed us to shed light on key steps of the catalytic cycle. Herein, we summarize the essential findings regarding the redox chemistry of [FeFe]-hydrogenases and discuss their catalytic hydrogen turnover. We furthermore will give an outlook on potential research activities and exploit the utilization of synthetic cofactor mimics.

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Section: Artificial Maturationsupporting

confidence: 70%

[FeFe]-Hydrogenases: recent developments and future perspectives

et al. 2018

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“…as well as differences in electron density distribution across the diiron site (PDS). 33 A notable spectroscopic feature is the lack of larger changes in the CNfrequencies among the COinhibited cofactor variants ( Fig. S2 and Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…1) affecting the geometry of the Fe d site, the reactivity with small molecules, and the hydrogen bonding network at the active site. [31][32][33] Such cofactor variants show characteristic infrared signatures, accumulate different H-cluster states in the presence of H 2 , and exhibit distinct CO sensitivities. [13][14][15] These observations prompted us to investigate the underlying substrate affinities and cofactor geometries.…”

Section: Introductionmentioning

confidence: 99%

Geometry of the Catalytic Active Site in [FeFe]-Hydrogenase Is Determined by Hydrogen Bonding and Proton Transfer

et al. 2019

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FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H 2. They serve as a blueprint for the design of synthetic H 2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H 2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H 2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H 2-forming catalysts.

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“…3). The behavior of TmHydA is reminiscent of that from Clostridium pasteurianum and Clostridium acetobutylicum [FeFe] hydrogenases both of which show a bias towards H + reduction at neutral pH [9,39].…”

Section: Catalytic Activity Using Protein Film Electrochemistrymentioning

confidence: 99%

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

et al. 2019

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The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H +) by dihydronicotinamide adenine dinucleotide (NADH) (∆G 0 ≈ 18 kJ mol −1) to the exergonic reduction of H + by reduced ferredoxin (Fd red) (∆G 0 ≈ − 16 kJ mol −1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H 2)-dependent reduction of nicotinamide adenine dinucleotide (NAD +) in the presence of oxidized ferredoxin (Fd ox) at a rate of ≈17 μmol NADH min −1 mg −1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H 2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.

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Chalcogenide substitution in the [2Fe] cluster of [FeFe]-hydrogenases conserves high enzymatic activity

Cited by 52 publications

References 59 publications

[FeFe]-Hydrogenases: recent developments and future perspectives

[FeFe]-Hydrogenases: recent developments and future perspectives

Geometry of the Catalytic Active Site in [FeFe]-Hydrogenase Is Determined by Hydrogen Bonding and Proton Transfer

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

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