SMALL-MOLECULE BINDING TO AN ARTIFICIALLY CREATED CAVITY AT THE ACTIVE-SITE OF CYTOCHROME-C PEROXIDASE

In the oxidized ''ES'' state of cytochrome c peroxidase, Trp-191 is re versibly oxidized to a stable cation free radical by the hypervalent h eme. To explore the potential for engineering a binding site for heter ocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-angstrom resol ution show that W191G contains a well-defined, approximately 180-angst rom3 cavity at the Trp-191 site. The cavity is occupied by five ordere d water molecules which participate in an extensive hydrogen-bonding n etwork with each other, with polar main-chain atoms, and with the carb oxylate of Asp-235. After a number of heterocyclic compounds were scre ened, evidence was obtained that substituted imidazoles bind to the ca vity of W191G. Titration of W191G with imidazole resulted in a perturb ation of the Soret absorption band that was not observed for W191H, W1 91F, or the native enzyme. The dissociation constants for binding of b enzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methyl imidazole, 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 steri c interactions and the efficiency of filling the cavity are important determinants for specificity. The K(d) 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 pr oton ionization curve with a pK(a) of 7.4, demonstrating the preferent ial binding by the imidazolium ion (pK(a) = 7.3). The binding of a num ber of substituted imidazoles to the cavity of W191G was verified by X -ray crystallographic analysis. The most clearly defined density was o bserved for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and wa s consistent with an oriented occupation in which the unsubstituted ni trogen forms a hydrogen bond or ion pair interaction with Asp-235. Thu s, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may st abilize the developing positive charge on the Trp-191 radical of the w ild-type enzyme. While the oxidation of imidazoles by the ferryl inter mediate of W191G was neither expected nor observed, this study has def ined the structural determinants for small molecule binding to an arti ficially created cavity near a heme center which is capable of generat ing oxidized species at a potential of over 1 V, and these results wil l guide future attempts for novel substrate oxidation by CCP.