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

被引：18

作者：

Ash, Philip A. ^{[1
]}

Reeve, Holly A. ^{[1
]}

Quinson, Jonathan ^{[1
]}

Hidalgo, Ricardo ^{[1
]}

Zhu, Tianze ^{[1
]}

McPherson, Ian J. ^{[1
]}

Chung, Min-Wen ^{[1
]}

Healy, Adam J. ^{[1
]}

Nayak, Simantini ^{[1
]}

Lonsdale, Thomas H. ^{[1
]}

Wehbe, Katia ^{[2
]}

Kelley, Chris S. ^{[2
]}

Frogley, Mark D. ^{[2
]}

Cinque, Gianfelice ^{[2
]}

Vincent, Kylie A. ^{[1
]}

机构：

[1] Univ Oxford, Dept Chem, Inorgan Chem Lab, S Parks Rd, Oxford OX1 3QR, England

[2] Diamond Light Source, Harwell Sci & Innovat Campus, Didcot OX11 0QX, Oxon, England

来源：

ANALYTICAL CHEMISTRY | 2016年 / 88卷 / 13期

基金：

英国生物技术与生命科学研究理事会; 欧洲研究理事会; 英国工程与自然科学研究理事会;

关键词：

SPECTROSCOPY; ENZYMES; HYDROGENASE; VOLTAMMETRY; ELECTRODES; FTIR; RADIATION; OXIDATION; PROTEINS; WATER;

D O I：

10.1021/acs.analchem.6b00898

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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 mononudeotide, 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 x 25 mu m(2). We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononudeotide 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.

引用

页码：6666 / 6671

页数：6

共 35 条

[11] Infrared Spectroscopy During Electrocatalytic Turnover Reveals the Ni-L Active Site State During H2 Oxidation by a NiFe Hydrogenase [J].

Hidalgo, Ricardo ;

Ash, Philip A. ;

Healy, Adam J. ;

Vincent, Kylie A. .

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2015, 54 (24) :7110-7113

[12] Opportunities for mesoporous nanocrystalline SnO2 electrodes in kinetic and catalytic analyses of redox proteins [J].

Kemp, Gemma L. ;

Marritt, Sophie J. ;

Li Xiaoe ;

Durrant, James R. ;

Cheesman, Myles R. ;

Butt, Julea N. .

BIOCHEMICAL SOCIETY TRANSACTIONS, 2009, 37 :368-372

[13] Voltage-dependent structural changes of the membrane-bound anion channel hVDAC1 probed by SEIRA and electrochemical impedance spectroscopy [J].

Kozuch, Jacek ;

Weichbrodt, Conrad ;

Millo, Diego ;

Giller, Karin ;

Becker, Stefan ;

Hildebrandt, Peter ;

Steinem, Claudia .

PHYSICAL CHEMISTRY CHEMICAL PHYSICS, 2014, 16 (20) :9546-9555

[14] Spectroelectrochemical Investigation of Intramolecular and Interfacial Electron-Transfer Rates Reveals Differences Between Nitrite Reductase at Rest and During Turnover [J].

Krzeminski, Lukasz ;

Ndamba, Lionel ;

Canters, Gerard W. ;

Aartsma, Thijs J. ;

Evans, Stephen D. ;

Jeuken, Lars J. C. .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2011, 133 (38) :15085-15093

[15] Enzyme electrokinetics:: Using protein film voltammetry to investigate redox enzymes and their mechanisms [J].

Léger, C ;

Elliott, SJ ;

Hoke, KR ;

Jeuken, LJC ;

Jones, AK ;

Armstrong, FA .

BIOCHEMISTRY, 2003, 42 (29) :8653-8662

[16] Direct electrochemistry of redox enzymes as a tool for mechanistic studies [J].

Leger, Christophe ;

Bertrand, Patrick .

CHEMICAL REVIEWS, 2008, 108 (07) :2379-2438

[17] Electric-field effects on the interfacial electron transfer and protein dynamics of cytochrome c [J].

Ly, H. Khoa ;

Wisitruangsakul, Nattawadee ;

Sezer, Murat ;

Feng, Jiu-Ju ;

Kranich, Anja ;

Weidinger, Inez M. ;

Zebger, Ingo ;

Murgida, Daniel H. ;

Hildebrandt, Peter .

JOURNAL OF ELECTROANALYTICAL CHEMISTRY, 2011, 660 (02) :367-376

[18] Protein voltammetry and spectroscopy: integrating approaches [J].

Male, Louise ;

Marritt, Sophie J. ;

Berks, Ben C. ;

Cheesman, Myles R. ;

van Wonderen, Jessica H. ;

George, Simon J. ;

Butt, Julea N. .

THEORETICAL CHEMISTRY ACCOUNTS, 2008, 119 (1-3) :107-111

[19]

Martin MC, 2013, NAT METHODS, V10, P861, DOI [10.1038/NMETH.2596, 10.1038/nmeth.2596]

[20]

Mattson E. C., 2014, INFRARED RAMAN SPECT, P585

← 1 2 3 4 →