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Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control

DOI: 10.1021/acs.analchem.6b00898 DOI Help

Authors: Philip A. Ash (Department of Chemistry, University of Oxford) , Holly Reeve (University of Oxford) , Jonathan Quinson (University of Oxford) , Ricardo Hidalgo (University of Oxford) , Tianze Zhu (University of Oxford) , Ian Mcpherson (University of Oxford) , Min-wen Chung (University of Oxford) , Adam Healy (University of Oxford) , Simantini Nayak (University of Oxford) , Thomas Lonsdale (University of Oxford) , Katia Wehbe (Diamond Light Source) , Chris S. Kelley (Diamond Light Source) , Mark Frogley (Diamond Light Source) , Gianfelice Cinque (Diamond Light Source) , Kylie Vincent (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Analytical Chemistry

State: Published (Approved)
Published: June 2016
Diamond Proposal Number(s): 9029

Abstract: We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10–1000 s–1, and therefore, fast experimental triggers are needed in order to study subturnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment, this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 μm2. We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control.

Subject Areas: Chemistry

Instruments: B22-Multimode InfraRed imaging And Microspectroscopy