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J. Biochem, 2003, Vol. 134, No. 6 859-867
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Reversely-Oriented Cytochrome b561 in Reconstituted Vesicles Catalyzes Transmembrane Electron Transfer and Supports Extravesicular Dopamine ß-Hydroxylase Activity

Yumiko Seike1, Fusako Takeuchi1 and Motonari Tsubaki*,1,2,3

1 Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-1297; 2 CREST, JST; and 3 Department of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501

Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine ß-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine ß-hydroxylase.

* To whom correspondence should be addressed. Tel/Fax: +81-78-803-6582, E-mail: mtsubaki{at}kobe-u.ac.jp


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N. Nakanishi, F. Takeuchi, and M. Tsubaki
Histidine Cycle Mechanism for the Concerted Proton/Electron Transfer from Ascorbate to the Cytosolic Haem b Centre of Cytochrome b561: A Unique Machinery for the Biological Transmembrane Electron Transfer
J. Biochem., November 1, 2007; 142(5): 553 - 560.
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