Substrates for microsomal azoreductase: Hammett substituent effects, NMR studies, and response to inhibitors

In previous studies on azoreduction by microsomal cytochrome P-450, we identified two classes of substrates structurally related to 4- dimethylaminoazobenzene. Both require polar electron-donating groups for binding to enzyme and are differentiated by their structure, their redox potentials, their rates of chemical and enzymic reduction, and the influence on their metabolism of inducing agents, CO and O₂. Azo compounds whose reductions are insensitive to CO and O₂ (I-substrates) contain electron- donating substituents on either ring. Azo compounds whose reductions are O₂- and CO-sensitive (S-substrates) also contain electron-withdrawing groups on the opposite (prime) ring. For all dyes, NMR studies revealed minor differences in the chemical shifts of the protons attached to the phenyl ring substituted with electron-donating substituents (ring A). This is consistent with the narrow range of pK(a)'s (basicity) and K(M) values for all substrates. However, there are significant differences in the chemical shifts of the aromatic protons of the prime ring (ring B). The difference in chemical shifts is most pronounced for aromatic protons adjacent to the prime ring substituents, showing a clear distinction between I and S substrates. Furthermore, the Hammett σ substituent constants on the prime ring clearly distinguish between the two classes of dyes. I- and S-substrates have negative and positive σ Hammett values, respectively. This implies that the mechanism of microsomal azoreduction is critically dependent on the charge and redox potentials of the dyes and is exclusively determined by the nature of the substituents on the prime ring. Inhibition of microsomal reduction by CN^- also distinguishes the two classes of dyes. Reduction of I-substrates is, on average, more sensitive to CN^- than is reduction of S-substrates. In contrast, inhibition by N₃^- does not distinguish I- from S-substrates. N₃^- acts exclusively on Fe³⁺ · P-450, whereas CN^- acts on both Fe²⁺ · and Fe³⁺ · P-450. Apparently, selective inhibition of Fe³⁺ · P-450 does not differentiate I- and S-substrates, whereas inhibition of Fe²⁺ · P-450 (CO, CN^-) does. In contrast to their lack of inhibition by CO, I- substrates are more sensitive to CN^-, possibly because of alteration of the enzyme redox potentials by CN^-. A mechanism for the inhibition by the heme- linked ligands is discussed.