Distinct roles of two heme centers for transmembrane electron transfer in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by pulse radiolysis.
The reaction of monodehydroascorbate (MDA) radical with purified cytochrome b561 from bovine adrenal chromaffin vesicles was investigated by the technique of pulse radiolysis. Radiolytically generated MDA radical oxidized rapidly the reduced form of cytochrome b561 to yield the oxidized form. Subsequently the oxidized form of cytochrome b561 was re-reduced by ascorbate in the medium. The second-order rate constants of the reaction of MDA radical were increased with decreasing pH, whereas a maximum of the second-order rate constant for the reaction with ascorbate was obtained around pH 6.8. At excess MDA radical to cytochrome b561 concentration, only half of the heme in cytochrome b561 was oxidized, indicating that only one of the two heme centers can react with MDA radical. On the other hand, when the reactions were examined using cytochrome b561 pretreated in a mild alkaline condition in the oxidized state, the cytochrome b561 could not be oxidized with MDA radical, suggesting that the heme center specific for the electron donation to MDA radical is selectively modified upon the alkaline treatment. These results suggest that the two heme b centers have distinct roles for the electron donation to MDA radical and the electron acceptance from ascorbate, respectively.
Kobayashi
K
K
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan. kobayasi@sanken.osaka-u.ac.jp
Tsubaki
M
M
Tagawa
S
S
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000201:enzymology*; D003573:Cytochrome b Group / Q000378:metabolism*; D003683:Dehydroascorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004579:Electron Transport; D005609:Free Radicals; D006418:Heme / Q000378:metabolism*; D011675:Pulse Radiolysis
D003573:Cytochrome b Group; D005609:Free Radicals; C023646:cytochrome b561; D006418:Heme; C000820:semidehydroascorbic acid; D003683:Dehydroascorbic Acid