Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c.
Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.
Waldmeyer
B
B
Bosshard
H R
HR
D000818:Animals; D002850:Chromatography, Gel; D003488:Cyanogen Bromide / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D006861:Hydrogen Peroxide / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010544:Peroxidases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
D003574:Cytochrome c Group; D010446:Peptide Fragments; D006861:Hydrogen Peroxide; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D003488:Cyanogen Bromide