Reaction of Horse Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I (original) (raw)

The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(M1) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-19 1 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 X lo8 M-' s-l. Ferrocytochrome c then reduces the oxyferryl heme Fe(1V) in CMPII with a rate constant of 2.0 X lo6 M-I s-I, The rate constant for the reduction of the radical is nearly independent of p H from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(1V) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(1V) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(M1). The intracomplex rate constants for reduction of the radical in these mutants by the rutheniumlabeled cytochrome c derivatives are also similar to that for CcP(M1). This rules out a direct role for these aromatic residues in electron transfer. These results support reduction of both the radical at Trp-191 and the oxyferryl heme Fe(1V) by the pathway recently proposed by Pelletier and Kraut [(1992) Science 258, 1748-17551 on the basis of the crystal structure of the complex between yeast CcP(M1) and yeast iso-1-cytochrome c. The cytochrome c-cytochrome c peroxidase system has been widely used for investigating fundamental questions about biological electron transfer. High-resolution crystallographic structures have been determined for both redox states of cytochrome c from a number of different organisms (Takano & Dickerson, 1981; Louie & Brayer, 1990). X-ray crystal structures have also been determined for yeast cytochrome c peroxidase in the resting, ferric state, CcP (Finzel et al., 1984), and the hydrogen peroxide oxidized state, CMPI (Edwards et al., 1987). In 1980, Poulos and Kraut proposed a hypothetical model for the 1:l complex between tuna cytochrome c and cytochrome c peroxidase that was based on the crystal structures of the individual proteins. This model complex is stabilized by charge-pair interactions between lysines 13, 27, 72, 86, and 87 surrounding the heme crevice of cytochrome c and the carboxylate groups on Asp-34,-37,-79, and-216 on cytochrome c peroxidase. Very recently, Pelletier and Kraut (1 992) determined the three-dimensional structure of a 1:l complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c crystallized at high ionic strength (150 mM NaC1, pH 7). The binding domain is different than that proposed in the Poulos-Kraut model, and hydrophobic and van der Waals interactions are particularly important in stabilizing the complex (Figure 1). No direct hydrogen bonds between the two proteins are present, This work was supported in part by NIH Grant GM20488 (to F.M. and B.D.) and NSF Grant MCB 9119292 (to J.K. and M.M.).