THE ROLE OF ASPARTATE-235 IN THE BINDING OF CATIONS TO AN ARTIFICIAL CAVITY AT THE RADICAL SITE OF CYTOCHROME-C PEROXIDASE

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically b ind substituted imidazoles in their protonated cationic forms (Fitzger ald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807- 3818). Proposals that a nearby carboxylate, Asp-235, and competing mon ovalent cations should modulate the affinity of the W191G cavity for l igand binding are addressed in this study. Competitive binding titrati ons of the imidazolium ion to W191G as a function of [K+] show that po tassium competes weakly with the binding of imidazoles. The dissociati on constant observed for potassium binding (18 mM) is more than 3,000- fold higher than that for 1,2-dimethylimidazole (5.5 mu M) in the abse nce of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unpert urbed by replacement of the carboxylate. Refined crystallographic B-va lues of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essentia l feature of the cation binding determinant and indicate that this car boxylate plays a critical role in stabilizing the formation of the Trp -191 radical cation.