Hydrazine and hydroxylamine as probes for O2-reduction site of mitochondrial cytochrome c oxidase.
Reactions of hydrazine and hydroxylamine with bovine heart cytochrome c oxidase in the fully reduced state were investigated under anaerobic conditions following the visible-Soret spectral change. Hydrazine gave a sharp band at 575 nm with 20% decrease in the alpha band at 603 nm, and hydroxylamine induced a 2 nm blue-shift for the alpha band without any clear splitting. The Soret band at 443 nm was decreased significantly in intensity, with the concomitant appearance of a shoulder with hydrazine or a peak with hydroxylamine, both near 430 nm. The dependence on pH of the affinity of these reagents for the enzyme indicates that only the deprotonated forms of these reagents bind to the enzyme, suggesting a highly hydrophobic environment of the haem ligand-biding site. These spectral changes were largely removed by addition of cyanide or CO. However, detailed analysis of these spectral changes indicates that hydrazine perturbs the shape of the spectral change induced by cyanide and hydroxylamine perturbs that induced by CO. These results suggest that these aldehyde reagents bind to haem a3 iron as well as to a second site which is most likely to be the formyl group on the haem periphery, and that these two sites bind these reagents anti-cooperatively with each other.
Kubota
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T
Department of Biology, Faculty of Science, Osaka University, Japan.
Yoshikawa
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S
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006834:Hydrazines / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry*; D007202:Indicators and Reagents; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D013057:Spectrum Analysis
D006834:Hydrazines; D006898:Hydroxylamines; D007202:Indicators and Reagents; C029424:hydrazine; D019811:Hydroxylamine; D003576:Electron Transport Complex IV; D010100:Oxygen