INFLUENCE OF MUTATION OF THE AMINO-TERMINAL SIGNAL ANCHOR SEQUENCE OFCYTOCHROME-P450 2B4 ON THE ENZYME STRUCTURE AND ELECTRON-TRANSFER PROCESSES

The role of the NH2-terminal hydrophobic patch of cytochrome P4502B4 ( CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) a nd cytochrome b(5) (b(5)) was assessed using a variant lacking the sig nal anchor sequence (Delta 2-27). CD, second-derivative, and fluoresce nce emission spectra indicated that the structure of the deletion muta nt slightly differed from that of the native CYP2B4, Fitting of the in itial-velocity patterns for P450R- and Es-directed electron transfer t o the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an app roximately 2.3-fold decrease in the affinity of the two electron donor s for the engineered enzyme, while the reductive efficiency remained u naffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Delta 2-27) accounted for this phe nomenon, interestingly, spectral docking of P450R to the truncated pig ment was not hampered, while the binding of b(5) was blocked. The rate s of sub strate-triggered aerobic NADPH consumption in systems contain ing CYP2B4(Delta 2-27) and P450R were 16 to 56% those obtained with th e unchanged hemoprotein, Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substra te-bound oxy complex. Experiments with b(5) as the ultimate electron d onor hinted at some damage to second-electron transfer to the truncate d enzyme. The results are consistent with the proposal that the NH2-te rminal hydrophobic region of CYP2B4 might be of importance in preserva tion of the catalytic competence of the enzyme.