Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay.
Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of oxidized cytochrome b(561) with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g(z) = 3.69 heme species was converted to a non-ascorbate-reducible form, although its g(z)-value showed no appreciable change. The treatment had no effect on the other heme (the g(z) = 3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b(561) when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-1297.
Fusako
F
Takeuchi
Hiroshi
H
Hori
Eiji
E
Obayashi
Yoshitsugu
Y
Shiro
Motonari
M
Tsubaki
D000818:Animals; D001205:Ascorbic Acid / Q000032:analysis / Q000378:metabolism*; D002417:Cattle; D019439:Chromaffin Cells / Q000737:chemistry* / Q000378:metabolism; D003573:Cytochrome b Group / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011199:Potentiometry; D013059:Spectrum Analysis, Raman / Q000379:methods*
D003573:Cytochrome b Group; C023646:cytochrome b561; D006418:Heme; D001205:Ascorbic Acid