The hepatic pigment accumulated in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated rats, which has been reported to inhibit ferrochelatase, has been isolated and purified. The pigment has been resolved into one major, one minor, and two trace components, all of which appear to be isomeric porphyrins. The major fraction has been unambiguously identified byspectroscopic methods as the isomer of N-methyprotoporphyrin IX (isolated as the dimethyl ester) in which vinyl-substituted pyrrole ring A is methylated. The minor product appears to be an isomer of the same porphyrin with the N-methyl group on propionic acid-substituted ring C, and the trace components have the same high-pressure liquid chromatography retention times as the other two possible isomers of the porphyrin. The four isomers of N-methylprotoporphyrin LX have been chemically synthesized, independently characterized, and used to confirm the structures of the biological products.The heuristic perturbation of hepatic heme synthesis by chemical agents has played a pivotal role in elucidation of the heme biosynthetic pathway and of the mechanism through which it is regulated. Two agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), have achieved particular prominence in this regard because the biosynthetic derangements that they engender mimic in many respects the inherited metabolic defects characteristic of certain human porphyrias (1). Administration of DDC to animals results in inhibition of the hepatic mitochondrial ferrochelatase responsible for the insertion of iron into the porphyrin ligand and consequently leads to the accumulation of protoporphyrin IX (2-4). Direct inhibition of ferrochelatase by DDC, however, appears not to be involved because the enzyme is not inhibited in vitro by this agent (2, 3). The mechanism of the inhibitory interaction has therefore remained obscure.More than a decade ago, two independent groups observed a rapid decrease in hepatic cytochrome P-450 levels in mice treated with either AIA or DDC (5, 6). Subsequent studies established that AIA-mediated loss of cytochrome P-450 reflected conversion of the enzyme prosthetic heme into an abnormal green pigment (7,8). We have established that the prosthetic heme is alkylated by an activated form of AIA produced during catalytic processing of this substrate, and we have demonstrated that the AIA green pigment is a covalent adduct of AIA with protoporphyrin IX (9-11). The interaction of DDC with cytochrome P-450, in contrast to the interaction of AIA with this enzyme, has not been intensively investigated. Tephly, Gibbs, and De Matteis, however, have recently reported that a green hepatic pigment is also formed in DDC-treated mice (12, 13) and have made the further important discovery that this pigment, unlike that obtained with AIA, is a potent inhibitor of mitochondrial ferrochelatase (13, 14). The differential physiological effects of AIA and DDC are thus related, at least in part, to differences in the structures of the accompa...