ABSTRACT
The function of cations in peroxidases is not entirely clear; however, the ions are thought to exert long-range electrostatic effects on the redox properties of the heme group. In the “ compound I” intermediate state of ascorbate peroxidase, one oxidizing equivalent exists in the form of a porphyrin 7r-cation radical [61]. In compound I of cytochrome c peroxidase, whose active site is very similar to that of ascorbate peroxidase, a cation radical on Trpl91 is present in compound I. Trpl91 is believed to provide an efficient route for electron transfer from cyto chrome c. The presence of the cation binding site, somewhat remote from the His-Asp-Trp triad, appears to be the major difference between the active sites of the two peroxidases. Hence, the cation was suspected of influencing the distribution of oxidizing equivalents and the stability of the compound I intermediate state in ascorbate peroxidase. Site-directed mutagenesis was used to engineer a cation bind ing site in cytochrome c peroxidase in a position analogous to that in ascorbate peroxidase. In order to create a K+ binding site, five amino acid substitutionsA176T, G192T, A194N, T199D and G201S-were inserted in the primary sequenceo [62]. A structure of the mutant enzyme at 1.5-A resolution confirmed the proper
folding of the variant and the presence of K+ in the engineered site [62]. The activity of the mutant form of cytochrome c peroxidase was inhibited by binding of K+ at the engineered site. The characteristic electron paramagnetic resonance signal for the compound I state was also suppressed when K+ was present. These results support the hypothesis that the remote cation in peroxidase has a profound influence on the redox properties of the heme group [61,62].
