Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b(561)

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorb ate. We found previously that treatment of oxidized cytochrome b(561) With diethyl pyrocarbonate caused specific N-carbethoxylation of three fully con served residues (His88, His161, and Lys85) located at the extravesicular si de. The modification lead to a selective loss of the electron-accepting abi lity from ascorbate without affecting the electron donation to monodehydroa scorbate radical [Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F., and Tagawa, S. (2000) Biochemistry 39, 3276-3284]. In the present study, we fo und that these modifications lead to a drastic decrease of the midpoint pot ential of heme b at the extravesicular side from +60 to -30 mV. We found fu rther that the O-carbethoxylation of one tyrosyl residue (Tyr218) located a t the extravesicular side was significantly enhanced under alkaline conditi ons, leading to a very slow reduction process of the oxidized heme b with a scorbate. On the other hand, the presence of ascorbate during the treatment with diethyl pyrocarbonate was found to suppress the carbethoxylation of H is88, His161, and Tyr218, whereas the modification level of Lys85 was not a ffected. Concomitantly, the final reduction level of heme b with ascorbate was protected, although the fast reduction phase was not fully restored. Th ese results suggest that the two heme-coordinating histidyl residues (His88 and His161) are also a part of the ascorbate binding site. Tyr218 and Lys8 5 may have a role in the recognition/binding process for ascorbate and are indispensable for the fast electron transfer reaction.