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

DOI: 10.1039/D1SC01734A DOI Help

Authors: Philip A. Ash (University of Oxford; University of Leicester) , Sophie E. T. Kendall-Price (University of Oxford) , Rhiannon M. Evans (University of Oxford) , Stephen Carr (University of Oxford; University of Leicester) , Amelia Brasnett (University of Oxford) , Simone Morra (University of Oxford; University of Nottingham) , Ricardo Hidalgo (University of Oxford) , Adam J. Healy (University of Oxford) , Gianfelice Cinque (Diamond Light Source; University of Oxford) , Mark D. Frogley (Diamond Light Source) , Fraser A Armstrong (University of Oxford) , Kylie A. Vincent (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemical Science

State: Published (Approved)
Published: June 2021
Diamond Proposal Number(s): 17753 , 19269 , 21651

Open Access Open Access

Abstract: Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials, Technique Development

Instruments: B22-Multimode InfraRed imaging And Microspectroscopy

Added On: 04/06/2021 10:53


Discipline Tags:

Physical Chemistry Biochemistry Technique Development - Chemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Spectroscopy Infrared Spectroscopy