A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver.

[14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.     

Kinetic studies of novel di- and tri-propionate substrates for the chicken red blood cell enzyme coproporphyrinogen oxidase

Coproporphyrinogen oxidase is an important enzyme in heme biosynthesis and catalyses the sequential oxidative decarboxylation of propionates on the A and B rings of the porphyrinogen ring. The effects of substituents on the C and D rings have not been systematically evaluated for their effects on the kinetic constants, K(m) and V(max). A series of synthetic porphyrinogens have been tested for their ability to affect these kinetic constants for the chicken enzyme. The enzyme exhibited the largest V(max) when incubated with the authentic substrate and was clearly able to distinguish between various substituents on the C and D rings of the macrocycle. When co-incubated with substrate, the authentic product, protoporphyrinogen-IX, appears to inhibit coproporphyrinogen oxidase and this may have an important role in the regulation of this enzyme. Thus the model for the active site of this enzyme should be modified to take these factors into account.     

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.     

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.     

Identification of a group of tetracarboxylate porphyrins, containing one acetate and three propionate β-substituents, in faeces from patients with symptomatic cutaneous hepatic porphyria and from rats with porphyria due to hexachlorobenzene

1. Three tetracarboxylate porphyrins, apart from coproporphyrin, were isolated from the faeces of patients with symptomatic cutaneous hepatic porphyria and of rats with porphyria caused by hexachlorobenzene poisoning. The porphyrins were not present in the urine of the porphyric rats or in the faeces of control rats. 2. Two of the porphyrins were identified as tris-(2-carboxyethyl)-carboxymethyl ethyl trimethyl porphin (isocoproporphyrin) and tris-(2-carboxyethyl)-carboxymethyl trimethyl porphin (de-ethyliso-coproporphyrin). 3. The third porphyrin, previously reported to be an analogue of coproporphyrin in which one propionate group is replaced by an alpha- or beta-hydroxypropionate group, was shown to be tris-(2-carboxyethyl)-carboxymethyl-(1-hydroxyethyl)-trimethyl porphin (hydroxyisocoproporphyrin). 4. The order of beta-substituents around the porphin ring could not be determined for any of the compounds. 5. Evidence for the existence of a fourth porphyrin, tris-(2-carboxyethyl)-carboxymethyl trimethyl vinyl porphin (dehydroisocoproporphyrin), a postulated precursor of the others, is described. 6. It is suggested that dehydroisocoproporphyrinogen is produced by decarboxylation-dehydrogenation of one of the propionate groups of pentacarboxylate porphyrinogen III.     

Multiple regulatory steps in erythroid heme biosynthesis

Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent down-stream rate-limiting steps.     

Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX.

The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis. Two previously isolated hemY mutations were sequenced. Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly. HemY was found to be a 53-kDa peripheral membrane-bound protein. In agreement with recent findings by Dailey et al. (J. Biol. Chem. 269:813-815, 1994) B. subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively. The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III. The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate. The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway. However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.     

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.     

Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.     

Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.     

The role of tyrosine 343 in substrate binding and catalysis by human sulfite oxidase

In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.     

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presence and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen IX to protoporphyrin IX. In the absence of herbicide, about 50% of the protoporphyrin IX formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period. In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid. When the incubation was carried out in the presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium. To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escherichia coli membranes. Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX. Protoporphyrin IX and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction. These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm. These results help explain the extraplastidic accumulation of protoporphyrin IX in plants treated with photobleaching herbicides. In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis.     

Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam)

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I. Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).     

Heme biosynthesis in human breast cancer--mimetic "in vitro" studies and some heme enzymic activity levels

1. Porphyrin biosynthesis from delta-aminolevulinic acid (ALA) was investigated using the technique of tissue explant cultures, in both human breast cancer and its original normal tissue. 2. The activity of ALA-dehydratase, porphobilinogenase and uroporphyrinogen decarboxylase was directly determined in both tumor and normal mammary tissues. 3. Porphyrin synthesis capacity of human breast carcinoma was 20-fold enhanced, as compared with normal tissue, at least between the stages of porphobilinogen and coproporphyrinogen formation. 4. The activity of the three enzymes examined was always lower in normal tissue than in tumoral tissue. 5. Present findings show that porphyrin biosynthesis is increased in breast cancer tissue.     

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.     

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein

Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants. The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX. The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Discovery of the true peroxy intermediate in the catalytic cycle of terminal oxidases by real-time measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O(2) to the fully reduced enzyme is followed by the fast (5 micros) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O(2) by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe (3+)(d)-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P(M) intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b(558) in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b(595) are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 micros; it decays into oxoferryl species due to oxidation of low spin heme b(558) that is linked to significant charge translocation across the membrane.     

Binding of protoporphyrin IX and metal derivatives to the active site of wild-type mouse ferrochelatase at low porphyrin-to-protein ratios

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.