Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control.pdf (984.83 kB)
Download file

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

Download (984.83 kB)
journal contribution
posted on 17.06.2019, 15:53 by PA Ash, HA Reeve, J Quinson, R Hidalgo, T Zhu, IJ McPherson, M-W Chung, AJ Healy, S Nayak, TH Lonsdale, K Wehbe, CS Kelley, MD Frogley, G Cinque, KA Vincent
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.

Funding

The work of K.A.V., P.A.A., H.A.R., and I.J.M. was supported by the European Research Council (EnergyBioCatalysis-ERC-2010-StG-258600), Engineering and Physical Sciences Research Council (EPSRC, EP/K031503/1, and INSPIRE award EP/J015202/1), and the Biotechnology and Biological Sciences Research Council (BB/L009722/1). H.A.R. was supported by a postdoctoral fellowship from the European Molecular Biology Organization. J.Q. and T.H.L. held EPSRC DTA awards EP/J500495/1 and EP/L505031/1, respectively. R.H. is supported by Ministerio de Ciencia y Tecnología, Universidad de Costa Rica, and Lincoln College, Oxford. M.-W.C. was supported by an Oxford Clarendon Fund Scholarship and A.J.H. by the John Fell Fund. S.N. holds a Marie Skłodowska-Curie actions individual fellowship (659306). We are grateful to Dr Alizé Pennec for assistance with isolation of NuoF and Prof. Luet Wong for access to molecular biology laboratories. We are grateful to Dr Miguel Ramirez for useful discussions. We thank Diamond Light Source for access to the MIRIAM beamline B22 (SM9029-1 and SM11611-1) that contributed to the results presented here.

History

Citation

Analytical Chemistry, 2016, 88 (13), pp. 6666-6671

Author affiliation

/Organisation/COLLEGE OF SCIENCE AND ENGINEERING/Department of Chemistry

Version

VoR (Version of Record)

Published in

Analytical Chemistry

Publisher

American Chemical Society

eissn

1520-6882

Acceptance date

07/06/2016

Copyright date

2016

Available date

17/06/2019

Publisher version

https://pubs.acs.org/doi/10.1021/acs.analchem.6b00898

Notes

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)

Language

en