Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. Nterminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the ␤␣␤ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the K m for protoporphyrinogen and the K i for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C-and N-terminal domains.Protoporphyrinogen oxidase (EC 1-3-3-4) is the penultimate enzyme of the heme biosynthesis pathway and the final enzyme of the common branch of the heme and chlorophyll biosynthesis pathways in plants. It catalyzes the oxidative O 2 -dependent aromatization of the colorless protoporphyrinogen IX to the highly conjugated protoporphyrin IX. Studies of the structure and function of protoporphyrinogen oxidase have been stimulated by the discovery that diphenyl ether-type herbicides are very potent inhibitors of protoporphyrinogen oxidase activity of yeast, mammal and plant mitochondria, and plant chloroplasts in vitro (1, 2). The phytotoxicity of diphenyl ether-type herbicides is lightdependent and involves intracellular peroxidation caused by protoporphyrin IX, the heme and chlorophyll precursor, leading to cell damage and lysis (3, 4) .The topological features at the active site of the protein must be studied if we are to understand the interactions of diphenyl ether-type herbicides with protoporphyrinogen oxid...

Protoporphyrinogen oxidase is a mitochondrial membranebound flavoprotein that catalyzes the penultimate reaction in the heme biosynthesis pathway, the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX (1). This enzyme has been shown to be the molecular target of diphenyl ether-type herbicides in plants (2, 3). The biochemical characteristics of the protein are those of an intrinsic protein, but sequence analysis of cloned yeast, mammalian, or plant protoporphyrinogen oxidases failed to reveal any typical membrane-spanning ␣-helical structure (4-9). Studies of the biogenesis of the yeast enzyme have shown that the protein is synthesized as a precursor that is rapidly converted to the active form of protoporphyrinogen oxidase, but that this maturation does not involve the removal of a N-terminal mitochondrial targeting peptide (6, 10). Although the sequence of protoporphyrinogen oxidase contains several hydrophobic regions, none of those is longer than 15 uncharged residues, and they are therefore unlikely to form membrane spanning segments; the largest hydrophobic domain in the protein is very homologous to the ␤␣␤-dinucleotide (Rossman)-binding fold found in many flavoproteins. Anchoring to the membrane may involve amphipathic helical domains that could be responsible for insertion of the protoporphyrinogen oxidase into the inner mitochondrial membrane, as described for prostaglandin H 2 synthase-1, a monotopic membrane-bound protein of the endoplasmic reticulum (11). The conserved domain A in protoporphyrinogen oxidases (12) (Fig. 1A) may well represent such an anchoring structure, and hydrophobic cluster analysis (13) of this domain (Fig. 1B) shows striking similarities to the membrane-anchoring domain of prostaglandin H 2 synthase (Fig. 1C). Another possibility, compatible with a posttranslational modification of the protein leading to the shift in electrophoretic mobility initially attributed to the proteolytic cleavage of a putative presequence previously described (10), is that protoporphyrinogen oxidase is anchored to the inner mitochondrial membrane by a different mechanism, such as acylation. This prompted us to look for a potential posttranslational modification of the polypeptide chain that could contribute to both the highly hydrophobic nature of the protein and the difference in electrophoretic mobility of the precursor and the mature forms of the protein. Studies on the biogenesis of the Ras oncogene products showed that acylation of these proteins may alter their electrophoretic mobility and hydrophobicity (14). We therefore investigated the possibility that protoporphyrinogen oxidase is an acylated protein. Materials and MethodsMaterials. Protoporphyrin IX, disodium salt, was purchased from Serva. l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (EC 3.4.21.4) from bovine pancreas used either as a soluble enzyme (Type XIII, 10,000-13,000 units⅐mg Ϫ1 ) or attached to agarose beads (75-100 units⅐ml Ϫ1 packed gel), cerulenin (2, 3-epoxy-4-oxo-7, 10 dodecadie...