Electrochemical and Spectroscopic Characteristics of Cytochrome P450 55A3 and Its Interaction with Nitric Oxide
International journal of biological macromolecules(2021)
摘要
Cytochrome P450 55A3 (CYP55A3) is an enzyme with the catalytic activity of nitric oxide (NO) to nitrous oxide using NADH or NADPH as the electron donor. Herein CYP55A3 has been expressed in E. coli and purified by His-tag columns. The electrochemical and spectroscopic characteristic of CYP55A3 and its interaction with NO has been studied. The direct electrochemistry of Fe3+/Fe2+ redox peaks in CYP55A3 was realized on the pyrolitic graphite electrode with the redox potential of -475 mV in pH 7.0 phosphate buffer. With the addition of NO a ferric nitroxyl complex (Fe3+-NO) formed with a new reduction peak at -0.78 V. The reduction peak current increased with the concentration of NO and showed typical Michaelis-Menten kinetic characteristics with the apparent Michaelis constant Kmapp 9.78 mu M. The binding constant K calculated to be 3.93 x 10(4) M by UV-vis method. The fluorescence emission spectra of iron porphyrin in CYP55A3 showed with the peak wavelength 633 nm, and its fluorescence intensity increased after binding with NO. The fluorescence analysis demonstrated that NADH can relay electrons to iron porphyrin and reduce NO. The reductive product of NO released and the iron porphyrin in CYP55A3 turned back to the original form. (C) 2020 Elsevier B.V. All rights reserved.
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关键词
Cytochrome P450 55A3,Nitric oxide,Electrochemical and spectrometric method
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