Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

Cornish, Adam J.; Ginovska, Bojana; Thelen, Adam; Silva, Júlio C. S. Da; Soares, Thereza A.; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J.; Hegg, Eric L.

doi:10.1021/acs.biochem.5b01044

Cited by 28 publications

(18 citation statements)

References 42 publications

(104 reference statements)

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“…Potentially involved amino acids have been identified due to phylogenetic conservation 82 and MD simulations. [18][19][20][21] However, cysteine 169 of the proton relay group at the cofactor (Fig. 2) is the only residue that has been verified to propel proton transfer to and from the H-cluster.…”

Section: Discussionmentioning

confidence: 99%

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[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: Discussionmentioning

confidence: 99%

“…Molecular dynamic (MD) simulations were employed to characterize this conserved proton transfer path. [18][19][20][21] It is formed by two glutamic acid side chains, at least one serine, one or two water molecules, and a cysteine in close vicinity to the adt group of the H-cluster (Fig. 2).…”

Section: This Geometry Stabilizes the Open Coordination Site At Fe D mentioning

confidence: 99%

[FeFe]-Hydrogenases: recent developments and future perspectives

et al. 2018

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“…[18][19][20]35 Theoretical studies support a rapid exchange of protons via this 'catalytic' pathway. [53][54][55] However, the interplay between proton transfer and electron transfer events in the hydrogen conversion reaction of [FeFe]hydrogenases remains largely unclear.…”

Section: Introductionmentioning

confidence: 99%

Protonation/reduction dynamics at the [4Fe–4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases

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Duan

et al. 2018

Phys. Chem. Chem. Phys.

345

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The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

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“…part of the W1/adt proton transfer pathway towards Fed. [8][9][10] Cysteine 417 at the cluster is a potential protonation site as part of proton transfer pathway W2.…”

Section: Introductionmentioning

confidence: 99%

The Molecular Proceedings of Biological Hydrogen Turnover

2018

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Over the past two decades, the bioinorganic chemistry of hydrogenases has attracted much interest from basic and applied research. Hydrogenases are highly efficient metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen (H) in all domains of life. Their iron- and nickel-based cofactors represent promising blueprints for the design of biomimetic, synthetic catalysts. In this Account, we address the molecular proceedings of hydrogen turnover with [FeFe]-hydrogenases. The active site cofactor of [FeFe]-hydrogenases ("H-cluster") comprises a unique diiron complex linked to a [4Fe-4S] cluster via a single cysteine. Since it was discovered that a synthetic analogue of the diiron site can be incorporated into apoprotein in vitro to yield active enzyme, significant progress has been made toward a comprehensive understanding of hydrogenase catalysis. The diiron site carries three to four carbon monoxide (CO) and two cyanide (CN) ligands that give rise to intense infrared (IR) absorption bands. These bands are sensitive reporters of the electron density across the H-cluster, which can be addressed by infrared spectroscopy to follow redox and protonation changes at the cofactor. Synthetic variation of the metal-bridging dithiolate ligand at the diiron site, as well as site-directed mutagenesis of amino acids, provides access to the proton pathways toward the cofactor. Quantum chemical calculations are employed to specifically assign IR bands to vibrational modes of the diatomic ligands and yield refined H-cluster geometries. Here, we provide an overview of recent research on [FeFe]-hydrogenases with emphasis on experimental and computational IR studies. We describe advances in attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) and protein film electrochemistry, as well as density functional theory (DFT) calculations. Key cofactor species are discussed in terms of molecular geometry, redox state, and protonation. Isotope editing is introduced as a tool to evaluate the cofactor geometry beyond the limits of protein crystallography. In particular, the role of proton-coupled electron transfer (PCET) in the generation of catalytically relevant redox species is addressed. We propose that site-selective protonation of the H-cluster biases surplus electrons either to the [4Fe-4S] cluster or to the diiron site. Protonation of the [4Fe-4S] cluster prevents premature reduction at the diiron site and stabilizes a reactive, terminal hydride. The observed H-cluster species are assigned to rapid H conversion or to reactions possibly involved in activity regulation and cellular H sensing. In the catalytic cycle of [FeFe]-hydrogenases, an H-cluster geometry is preserved that features a bridging CO ligand. PCET levels the redox potential for two steps of sequential cofactor reduction. The concept of consecutive PCET at a geometrically inert cofactor with tight control of electron and proton localization may inspire the design of a novel generation of biomimetic catalysts for the pro...

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Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

Cited by 28 publications

References 42 publications

[FeFe]-Hydrogenases: recent developments and future perspectives

[FeFe]-Hydrogenases: recent developments and future perspectives

Protonation/reduction dynamics at the [4Fe–4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases

The Molecular Proceedings of Biological Hydrogen Turnover

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