Please use this identifier to cite or link to this item:
Title: Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control.
Authors: Ash, PA
Reeve, HA
Quinson, J
Hidalgo, R
Zhu, T
McPherson, IJ
Chung, M-W
Healy, AJ
Nayak, S
Lonsdale, TH
Wehbe, K
Kelley, CS
Frogley, MD
Cinque, G
Vincent, KA
First Published: 7-Jun-2016
Publisher: American Chemical Society
Citation: Analytical Chemistry, 2016, 88 (13), pp. 6666-6671
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 μm(2). 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.
DOI Link: 10.1021/acs.analchem.6b00898
eISSN: 1520-6882
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © the authors, 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Description: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00898. Additional experimental data, including details of the preparation of cell-free extract of NuoF subunit of E. coli complex I; scale diagram of the microspectroscopy cell; comparison of electrochemical response of the microspectroscopy and ATR-IR cells; image of the particle-modified electrode used to record data presented in Figure 4; difference spectra showing the reproducibility of spectral data in the microspectroscopic cell; NuoF CE difference spectrum compared to the baseline noise level; absence of flavin signal in cell-free extract deficient in NuoF; absence of electrocatalytic NAD+NADH cycling in cell-free extract deficient in NuoF (PDF) pdf ac6b00898_si_001.pdf (710.75 kb)
Appears in Collections:Published Articles, Dept. of Chemistry

Files in This Item:
File Description SizeFormat 
Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control.pdfPublished (publisher PDF)984.83 kBAdobe PDFView/Open

Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.