Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

Morra, Simone; Duan, Jifu; Winkler, Martin; Ash, Philip A.; Happe, Thomas; Vincent, Kylie A.

doi:10.1039/d1dt02219a

Cited by 11 publications

(16 citation statements)

References 52 publications

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“…The method reported here, already extended to crystals of C. pasteurianum [FeFe] hydrogenase I, 78 is likely to have general relevance in structure–function studies of complex redox metalloenzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Significantly, we now show that, within single crystals of Hyd1, it is possible to achieve control over the full manifold of states observed in solution for this enzyme. A related report by Morra et al demonstrates electrochemical manipulation of [FeFe] hydrogenase I from Clostridium pasteurianum using similar methods, 78 and these studies present the possibility for using electrochemical control over single protein crystals to establish samples in the solid state for further structural study.…”

Section: Introductionmentioning

confidence: 95%

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The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

et al. 2021

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

et al. 2021

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“…Furthermore, H inact could be reached by (electro)chemical oxidation in the absence of O 2 . Recently, the Vincent group demonstrated that this approach could be extended to study individual crystals of [FeFe]-hydrogenase from Clostridium pasteurianum (“CpI”), using a cocktail of electron mediators to shuttle electrons and ultimately control the redox state of the enzyme during FTIR microspectroscopy . Importantly, this approach allowed all of the catalytically relevant states of CpI to be probed, in addition to identifying a previously undetected catalytic state ( H redH + ).…”

Section: Methods To Study Enzymatic and Microbial Electrodesmentioning

confidence: 99%

Enzymatic and Microbial Electrochemistry: Approaches and Methods

et al. 2022

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The coupling of enzymes and/or intact bacteria with electrodes has been vastly investigated due to the wide range of existing applications. These span from biomedical and biosensing to energy production purposes and bioelectrosynthesis, whether for theoretical research or pure applied industrial processes. Both enzymes and bacteria offer a potential biotechnological alternative to noble/rare metal-dependent catalytic processes. However, when developing these biohybrid electrochemical systems, it is of the utmost importance to investigate how the approaches utilized to couple biocatalysts and electrodes influence the resulting bioelectrocatalytic response. Accordingly, this tutorial review starts by recalling some basic principles and applications of bioelectrochemistry, presenting the electrode and/or biocatalyst modifications that facilitate the interaction between the biotic and abiotic components of bioelectrochemical systems. Focus is then directed toward the methods used to evaluate the effectiveness of enzyme/bacteria−electrode interaction and the insights that they provide. The basic concepts of electrochemical methods widely employed in enzymatic and microbial electrochemistry, such as amperometry and voltammetry, are initially presented to later focus on various complementary methods such as spectroelectrochemistry, fluorescence spectroscopy and microscopy, and surface analytical/characterization techniques such as quartz crystal microbalance and atomic force microscopy. The tutorial review is thus aimed at students and graduate students approaching the field of enzymatic and microbial electrochemistry, while also providing a critical and up-to-date reference for senior researchers working in the field.

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“…Additionally, RR and IR spectroscopic studies were successfully performed on the same single hydrogenase crystals, complementing the crystallographic data and facilitating structural assignment of ambiguous inhibited and active redox states, such as the oxygen-stable H inact and the hydrogen-binding intermediate Ni a -S state in [FeFe] and [NiFe] hydrogenases, respectively [86,89]. Notably, well-defined redox states in hydrogenase single crystals for IR imaging have been generated by electrochemical and gas control [90][91][92][93]. Furthermore, Fe-OH ligation at the unprecedented [4Fe-3S] cluster of the O 2 -tolerant membrane-bound [NiFe] hydrogenase from R. eutropha has been identified by RR spectroscopy and confirmed by theoretical quantum and molecular mechanics methods [94,95].…”

Section: Hydrogenasesmentioning

confidence: 99%

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies

et al. 2021

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Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN− ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.

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Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

Abstract: Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and...

Cited by 11 publications

References 52 publications

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

Enzymatic and Microbial Electrochemistry: Approaches and Methods

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies

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