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

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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 cleav-age 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 β-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 β-hydroxylase.