Evidence for the stereoselective inhibition of chick embryo hepatic ferrochelatase by N-alkylated porphyrins. II.

N-Ethylprotoporphyrin (N-ethyl-PP) was isolated from the livers of phenobarbital-pretreated rats after the administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine, separated into its four regioisomers by high performance liquid chromatography, and quantitated. The percentage ratio, in the chromatogram, of the peak areas of the ring A-substituted (NA) and the ring B-substituted (NB) regioisomers was 80:20, compared with 50:50 for synthetic N-ethyl-PP. The NA regioisomer of N-ethyl-PP isolated from rat liver was found to be approximately 5 times more potent an inhibitor of ferrochelatase than was the NB regioisomer. Because the synthetic NA regioisomer (an equal mixture of the NA and the epi-NA enantiomers) is equipotent with the synthetic NB regioisomer (an equal mixture of the NB and the epi-NB enantiomers), epi-NB must be more potent than epi-NA. The higher potency previously observed with the NA plus NB regioisomers of N-ethyl-PP isolated from rat liver, compared with the NA plus NB regioisomers of synthetic N-ethyl-PP, is explained by the fact that the biological preparation contains 80% of the potent NA, compared with 25% of the potent NA and 25% of the potent epi-NB in the synthetic preparation. The critical features for optimal ferrochelatase-inhibitory activity are the ring A N-ethyl group facing downward in the NA isomer and the ring B N-ethyl group in the epi-NB isomer being rotated through 180 degrees to occupy the same position. According to one proposed mechanism, N-alkylprotoporphyrins inhibit ferrochelatase by serving as transition state analogues for the iron insertion step. X-ray crystallography shows that the N-alkyl group-bearing pyrrole ring and the pyrrole ring opposite to the N-alkyl group are tilted out of planarity in opposite directions. We suggest that this tilting reflects the normal conformational changes required for the insertion of iron into the protoporphyrin IX ring by ferrochelatase and that the greater inhibitory activity of NA and epi-NB isomers, compared with epi-NA and NB isomers, is due to the fact that the normal mechanism for ferrochelatase-catalyzed iron insertion has preference for an A-C ring tilt over a B-D ring tilt.