TOPOLOGICAL MAPPING OF THE ACTIVE-SITES OF CYTOCHROME-P4502B1 AND CYTOCHROME-P4502B2 BY INSITU REARRANGEMENT OF ARYL-IRON COMPLEXES

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (N(A)) and D (N(D)) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-arylprotoporphyrin IX N(A):N(D) regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (<2:>98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the N(D) regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the N(A) regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A. An aryl-iron complex is formed with 4-(4-hydrazinophenyl)benzenesulfonic acid, but the aryl ligand appears to be too tall in this instance to shift toward either porphyrin nitrogen. Substrates bind primarily over pyrrole ring D because binding of benzphetamine, aminopyrine, or testosterone to the phenyl-iron complex before the oxidative phenyl shift increases the proportion of the N(A) regioisomer. Similar results are obtained with cytochrome P4502B2. A refined topological model based on these results is suggested for the active sites of cytochromes P4502B1 and 2B2.