Effects of reversing the protein positive charge in the proximity of the flavin N(1) locus of choline oxidase

Biochemistry
Mahmoud Ghanem, Giovanni Gadda

Abstract

A protein positive charge near the flavin N(1) locus is a distinguishing feature of most flavoprotein oxidases, with mechanistic implications for the modulation of flavin reactivity. A recent study showed that in the active site of choline oxidase the protein positive charge is provided by His(466). Here, we have reversed the charge by substitution with aspartate (CHO-H466D) and, for the first time, characterized a flavoprotein oxidase with a negative charge near the flavin N(1) locus. CHO-H466D formed a stable complex with choline but lost the ability to oxidize the substrate. In contrast to the wild-type enzyme, which binds FAD covalently in a 1:1 ratio, CHO-H466D contained approximately 0.3 FAD per protein, of which 75% was not covalently bound to the enzyme. Anaerobic reduction of CHO-H466D resulted in the formation of a neutral hydroquinone, with no stabilization of the flavin semiquinone; in contrast, the anionic semiquinone and hydroquinone species were observed with the wild type and a H466A variant of the enzyme. The midpoint reduction potential for the oxidized-reduced couple in CHO-H466D was approximately 160 mV lower than that of the wild-type enzyme. Finally, CHO-H466D lost the ability to form complexes with glycin...Continue Reading

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Citations

Jan 30, 2013·Biochemistry·Methinee ProngjitPimchai Chaiyen
Feb 26, 2008·Proceedings of the National Academy of Sciences of the United States of America·Joanne I YehShoucheng Du
Apr 29, 2009·The Journal of Biological Chemistry·Osbourne QuayeGiovanni Gadda
Apr 27, 2010·Archives of Biochemistry and Biophysics·Michelle Henderson Pozzi, Paul F Fitzpatrick
Dec 7, 2007·Chemistry : a European Journal·Jirí SvobodaBurkhard König
Jun 22, 2010·Archives of Biochemistry and Biophysics·Steffan FinneganGiovanni Gadda
Jan 27, 2007·Journal of Enzyme Inhibition and Medicinal Chemistry·Ozden Tacal, Inci Ozer
Feb 26, 2016·PloS One·Christian KochRalf Ficner
Feb 15, 2007·The Journal of Biological Chemistry·Alex BerndtEva Wolf
Oct 17, 2017·Archives of Biochemistry and Biophysics·Giovanni Gadda, Hongling Yuan
Jun 11, 2008·Biochemistry·Kunchala Rungsrisuriyachai, Giovanni Gadda
Feb 27, 2010·Biochemistry·Steffan FinneganGiovanni Gadda
Dec 6, 2014·Biochemistry·Crystal SmithermanGiovanni Gadda

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