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Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli

DOI: 10.1039/D2SC05641K DOI Help

Authors: Rhiannon M. Evans (University of Oxford) , Stephen E. Beaton (University of Oxford) , Patricia Rodriguez Macia (University of Oxford) , Yunjie Pang (Beijing Normal University; Max Planck Institute for Chemical Energy Conversion) , Kin Long Wong (University of Oxford; Research Complex at Harwell) , Leonie Kertess (University of Oxford) , William K. Myers (University of Oxford) , Ragnar Bjornsson (Max Planck Institute for Chemical Energy Conversion; Univ Grenoble Alpes, CNRS, CEA) , Philip A. Ash (The University of Leicester) , Kylie A. Vincent (University of Oxford) , Stephen B. Carr (University of Oxford; Research Complex at Harwell) , Fraser A. Armstrong (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemical Science , VOL 108

State: Published (Approved)
Published: July 2023
Diamond Proposal Number(s): 12346 , 21651

Open Access Open Access

Abstract: The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: B22-Multimode InfraRed imaging And Microspectroscopy , I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 01/08/2023 09:28


Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Spectroscopy Macromolecular Crystallography (MX) Infrared Spectroscopy Synchtron-based Fourier Transform Infrared Spectroscopy (SR-FTIR)