Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis.
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 ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.
Tsubaki
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-1297, Japan. tsubaki@sci.himeji-tech.ac.jp
Kobayashi
K
K
Ichise
T
T
Takeuchi
F
F
Tagawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry*; D002417:Cattle; D003573:Cytochrome b Group / Q000737:chemistry*; D003683:Dehydroascorbic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004579:Electron Transport; D005609:Free Radicals / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011675:Pulse Radiolysis; D012697:Serine Endopeptidases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003573:Cytochrome b Group; D005609:Free Radicals; D005960:Glucosides; D010446:Peptide Fragments; C023646:cytochrome b561; C018619:octyl-beta-D-glucoside; C000820:semidehydroascorbic acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D004047:Diethyl Pyrocarbonate; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid