Existence of two heme B centers in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy.
We have established a new purification procedure of cytochrome b561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at gz = 3.14 and gz = 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at gz = 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (bL or b566) of the mitochondrial complex III, indicating that the gz = 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid- and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.
Tsubaki
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-12, Japan. tsubaki@sci/himeji-tech.ac.jp
Nakayama
M
M
Okuyama
E
E
Ichikawa
Y
Y
Hori
H
H
D000313:Adrenal Medulla / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000737:chemistry*; D003573:Cytochrome b Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003573:Cytochrome b Group; C023646:cytochrome b561; D006418:Heme