New insights into the oxidation process from neutron and X-ray crystal structures of an O<sub>2</sub>-sensitive [NiFe]-hydrogenase

Hiromoto, Takeshi; Nishikawa, Koji; Inoue, Seiya; Ogata, Hideaki; Hori, Yuta; Kusaka, Katsuhiro; Hirano, Yu; Kurihara, Kazuo; Shigeta, Yasuteru; Tamada, Taro; Higuchi, Yoshiki

doi:10.1039/d3sc02156d

Cited by 5 publications

(3 citation statements)

References 57 publications

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“…Crucially, we report structural details of intermediates involved in the removal of protons from the [NiFe] active site during catalytic H2 oxidation. These results resolve the knowledge-gap between existing reported structures (in which there are no amino acid residues within hydrogen-bonding distance of the active site) 18,19 and current kinetic studies 20,21 that suggest involvement of proton tunnelling (requiring a hydrogen-bonding connection of no more than 2.7 Å). Our results allow us to present a model for how a finely-tuned redox enzyme combines the active site rigidity required for rapid electron transfer with a state-specific conformational flick that enables proton transfer to keep pace with catalytic turnover.…”

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confidence: 77%

“…4) isolates the Ni-B state with each structure showing clear electron density for a hydroxide bridging the Ni and Fe atoms. 19 Unlike air-oxidation of Hyd-1 there is no indication of oxygenation of any of the active site cysteine residues, indicative of Ni-A. 44 At +100 mV the proximal cluster remains in the closed conformation 45 with the mobile Fe atom (Fe7) coordinated by the side-chains of C19 and C20 (Extended Data Fig 9).…”

Section: Fine Potential Control Uncouples Structural Changes At the A...mentioning

confidence: 99%

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Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Carr,

Li,

Wong

et al. 2024

Preprint

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Redox enzymes capable of carrying out multi-electron reactions serve as blueprints for the rational design of bio-inspired catalysts for future green technologies. During catalysis, enzymes transition through multiple ‘active’ intermediate states. Movement of both electrons and substrate to/from the active site is precisely controlled to achieve the equivalent of high Faradaic efficiencies, with minimal electrons wasted in detrimental side reactions. High turnover frequencies in metalloenzymes such as the nickel-iron (NiFe) hydrogenases require mechanisms for highly-choreographed movement of two quantum particles, protons and electrons. According to Marcus theory, structural rigidity is key to a low reorganisation energy barrier for rapid, outer-sphere electron transfer. However, this is at odds with the requirement for a degree of conformational flexibility to enable rapid proton tunnelling between residues separated by distances greater than ~2.7 Å. Here we exploit the specific redox poising possible with electrochemical control of protein crystals and characterise, structurally and spectroscopically, [NiFe]-hydrogenase in each of the key states of the catalytic cycle as well as its CO-bound and oxidised states. These structures confirm extraordinarily fixed metal coordination at the active site, conducive to fast multi-electron catalysis, and reveal a subtle carboxylate ‘flick’ that provides molecular detail of how glutamate acts as a proton shuttle.

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confidence: 77%

Section: Fine Potential Control Uncouples Structural Changes At the A...mentioning

confidence: 99%

Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Carr,

Li,

Wong

et al. 2024

Preprint

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“…Neutron crystallography that can visualize H atoms is an extremely useful technique for that purpose. Structural details of NH–S bonds with ligand atoms of metalloproteins have only recently started to be uncovered by neutron and sub-ångström resolution X-ray crystallography, − whereas it is well-known that they control redox potentials. The presence of the NH–S bond at metal centers of various metalloproteins suggests that it is one of nature’s favorite strategies to tune the metalloprotein activity.…”

Section: Discussionmentioning

confidence: 99%

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography

Fukuda,

Lintuluoto,

Kurihara

et al. 2024

Biochemistry

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Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure−function relationship. We here used neutron and sub-ångstrom resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Åresolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-ångstrom resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

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Reactivation and long-term stabilization of the [NiFe] Hox hydrogenase of Synechocystis sp. PCC6803 by glutathione after oxygen exposure

Romig,

Eberwein,

Deobald

et al. 2024

Journal of Biological Chemistry

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New insights into the oxidation process from neutron and X-ray crystal structures of an O₂-sensitive [NiFe]-hydrogenase

Abstract: We report the first neutron structure of [NiFe]-hydrogenase in its oxidized state. This study leads to new insights into the oxidized active site and visualization of the protons characteristic of the oxidized enzyme.

Cited by 5 publications

References 57 publications

Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography

Reactivation and long-term stabilization of the [NiFe] Hox hydrogenase of Synechocystis sp. PCC6803 by glutathione after oxygen exposure

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New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase

Abstract: We report the first neutron structure of [NiFe]-hydrogenase in its oxidized state. This study leads to new insights into the oxidized active site and visualization of the protons characteristic of the oxidized enzyme.

Cited by 5 publications

References 57 publications

Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Glutamate “flick” enables proton tunneling during fast redox biocatalysis

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography

Reactivation and long-term stabilization of the [NiFe] Hox hydrogenase of Synechocystis sp. PCC6803 by glutathione after oxygen exposure

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Product

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New insights into the oxidation process from neutron and X-ray crystal structures of an O₂-sensitive [NiFe]-hydrogenase