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Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal.

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posted on 2019-06-18, 08:08 authored by PA Ash, SB Carr, HA Reeve, A Skorupskaitė, JS Rowbotham, R Shutt, MD Frogley, RM Evans, G Cinque, FA Armstrong, KA Vincent
We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.


The work of FAA, SBC, RME and KAV is supported by BBSRC grants BB/L009722/1 and BB/N006321/1. Work of KAV, PAA, HAR and JSR is additionally supported by EPSRC grant EP/N013514/1. FAA is a Royal Society Wolfson Research Merit Award holder. AS is grateful for a scholarship from the Oxford Interdisciplinary Bioscience Doctoral Training Partnership, BB/M011224/1. We thank Diamond Light Source for access to the MIRIAM beamline B22 (SM13879) that contributed to the results presented here. We also thank Elena Nomerotskaia for assistance with isolation of Hyd1.



Chemical Communications, 2017, 53 (43), pp. 5858-5861

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Royal Society of Chemistry



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Electronic supplementary information (ESI) available: Crystal preparation; cyclic voltammogram in the experimental cell; position-dependent difference spectra; demonstration of control over all active sites; visible images of a crystal recorded periodically during measurements; time-dependent Nia-R formation. See DOI: 10.1039/c7cc02591b