Quinone Electrophiles Selectively Adduct “Electrophile Binding Motifs” within Cytochrome c

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Abstract
Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein−protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile−protein adducts produce structural alterations that influence biological function.