Selective perturbation of the intravesicular heme center of cytochrome b561 by cysteinyl modification with 4,4'-dithiodipyridine.
Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at g(z) = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of purified cytochrome b(561) in an oxidized state with a sulfhydryl reagent, 4,4'-dithiodipyridine, caused the introduction of only one 4-thiopyridine group per b(561) molecule at either Cys57 or Cys125. About half of the heme centers of the modified cytochrome were reduced rapidly with ascorbate as found for the untreated sample, but the final reduction level decreased to approximately 65%. EPR spectra of the modified cytochrome showed that a part of the g(z) = 3.14 low-spin EPR species was converted to a new low-spin species with g(z) = 2.94, although a considerable part of the heme center was concomitantly converted to a high-spin g = 6 species. Addition of ascorbate to the modified cytochrome caused the disappearance or significant reduction of the EPR signals at g(z) = 3.69 and 3.14 of low-spin species and at g = 6.0 of the high-spin species, but not for the g(z) approximately 2.94 species. These results suggested that the bound 4-thiopyridone at either Cys57 or Cys125 affected the intravesicular heme center and converted it partially to a non-ascorbate-reducible form. The present observations suggested the importance of the two well-conserved Cys residues near the intravesicular heme center and implied their physiological roles during the electron donation to the monodehydroascorbate radical.
Takeuchi
Fusako
F
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501.
Hori
Hiroshi
H
Tsubaki
Motonari
M
D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules; D003545:Cysteine / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D011725:Pyridines / Q000737:chemistry*; D013439:Sulfhydryl Reagents
D003573:Cytochrome b Group; D004220:Disulfides; D011725:Pyridines; D013439:Sulfhydryl Reagents; C023646:cytochrome b561; C007542:4,4'-dipyridyl disulfide; D003545:Cysteine