J. Biochem, 2004, Vol. 135, No. 1 53-64
© 2004 The Japanese Biochemical Society
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Properties of Two Distinct Heme Centers of Cytochrome b561 from Bovine Chromaffin Vesicles Studied by EPR, Resonance Raman, and Ascorbate Reduction Assay
,1,2,51 Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-1297; 2 Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501; 3 Division of Biophysical Engineering, Department of Systems and Human Science, Graduate School of Engineering Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-8531; 4 Biophysical Chemistry Laboratory, RIKEN, Harima Institute, Mikazuki-cho, Sayo-gun, Hyogo 679-5148; and 5 CREST, JST.
Cytochrome b561 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 ß-hydroxylase. Treatment of oxidized cytochrome b561 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 gz = 3.69 heme species was converted to a non-ascorbate-reducible form, although its gz-value showed no appreciable change. The treatment had no effect on the other heme (the gz = 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 b561 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.
* Present address: MRC Laboratory of Molecular Biology, Structural Division, Hills Road, Cambridge CB2 2QH, UK.
To whom corresponding should be addressed. Tel/Fax: +81-78-803-6582, E-mail: mtsubaki{at}kobe-u.ac.jp
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