THE REACTION OF ESCHERICHIA-COLI CYTOCHROME BO WITH H2O2 - EVIDENCE FOR THE FORMATION OF AN OXYFERRYL SPECIES BY 2 DISTINCT ROUTES

We have re-examined the reaction of fast oxidised cytochrome bo with H 2O2 in a stopped-flow spectrophotometer. Monitoring the reaction at 58 2 nm allows us to observe the formation and decay of a spectroscopical ly distinct intermediate which accumulates transiently prior to the fo rmation of an oxyferryl species previously characterised in this labor atory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469-475 [1]). The reaction shows three distin ct phases of which the fast and intermediate phases are bimolecular an d show a marked pH dependence. Initially these results appeared incomp atible with the report that only one equivalent of H2O2 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) fur . J. Biochem. 226, 731-737 [2]). However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.