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In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly
oxidized to a stable cation free radical by the hypervalent heme. To explore the
potential for engineering a binding site for heterocyclic compounds at this
site, the mutant W191G was constructed. Two independent crystal structures of
W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined,
approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five
ordered water molecules which participate in an extensive hydrogen-bonding
network with each other, with polar main-chain atoms, and with the carboxylate
of Asp-235. After a number of heterocyclic compounds were screened, evidence was
obtained that substituted imidazoles bind to the cavity of W191G. Titration of
W191G with imidazole resulted in a perturbation of the Soret absorption band
that was not observed for W191H, W191F, or the native enzyme. The dissociation
constants for binding of benzimidazole, imidazole, 2-ethylimidazole,
1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were
respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest
binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric
interactions and the efficiency of filling the cavity are important determinants
for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to
3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa
of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa =
7.3). The binding of a number of substituted imidazoles to the cavity of W191G
was verified by X-ray crystallographic analysis. The most clearly defined
density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and
was consistent with an oriented occupation in which the unsubstituted nitrogen
forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced
binding of positively charged molecules may be the result of interactions with
this carboxylate. An analogous interaction may stabilize the developing positive
charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of
imidazoles by the ferryl intermediate of W191G was neither expected nor
observed, this study has defined the structural determinants for small molecule
binding to an artificially created cavity near a heme center which is capable of
generating oxidized species at a potential of over 1 V, and these results will
guide future attempts for novel substrate oxidation by CCP.
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