© 2005 The Japanese Biochemical Society.
Regular Paper |
Selective Perturbation of the Intravesicular Heme Center of Cytochrome b561 by Cysteinyl Modification with 4,4'-Dithiodipyridine
1Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501; 2 Division of Bioengineering, Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-8531; and 3 CREST, JST
* To whom correspondence should be addressed. E-mail: mtsubaki{at}kobe-u.ac.jp
Cytochrome b561 from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at gz = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine ß-hydroxylase. Treatment of purified cytochrome b561 in an oxidized state with a sulfhydryl reagent, 4,4'-dithiodipyridine, caused the introduction of only one 4-thiopyridine group per b561 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 
65%. EPR spectra of the modified cytochrome showed that a part of the gz = 3.14 low-spin EPR species was converted to a new low-spin species with gz = 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 gz = 3.69 and 3.14 of low-spin species and at g = 6.0 of the high-spin species, but not for the gz 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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  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. [Abstract] [Full Text] [PDF]
|
|