An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

Toward a structome of Acinetobacter baumannii drug targets.

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii.

Recent advances in HemN-like radical S-adenosyl-l-methionine enzyme-catalyzed reactions.

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp3 carbon or add onto an sp2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.

Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in Tetrapyrrole Pathways.

Microorganisms have lifestyles and metabolism adapted to environmental niches, which can be very broad or highly restricted. Molecular oxygen (O2) is currently variably present in microenvironments and has driven adaptation and microbial differentiation over the course of evolution on Earth. Obligate anaerobes use enzymes and cofactors susceptible to low levels of O2 and are restricted to O2-free environments, whereas aerobes typically take advantage of O2 as a reactant in many biochemical pathways and may require O2 for essential biochemical reactions. In this Perspective, we focus on analogous enzymes found in tetrapyrrole biosynthesis, modification, and degradation that are catalyzed by O2-sensitive radical S-adenosylmethionine (SAM) enzymes and by O2-dependent metalloenzymes. We showcase four transformations for which aerobic organisms use O2 as a cosubstrate but anaerobic organisms do not. These reactions include oxidative decarboxylation, methyl and methylene oxidation, ring formation, and ring cleavage. Furthermore, we highlight biochemically uncharacterized enzymes implicated in reactions that resemble those catalyzed by the parallel aerobic and anaerobic enzymes. Intriguingly, several of these reactions require insertion of an oxygen atom into the substrate, which in aerobic enzymes is facilitated by activation of O2 but in anaerobic organisms requires an alternative mechanism.

The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase.

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.

The enzyme engineering of mutant homodimer and heterodimer of coproporphyinogen oxidase contributes to new insight into hereditary coproporphyria and harderoporphyria.

Hereditary coproporphyria (HCP) is an autosomal dominant-inherited disease of haem biosynthesis caused by partial deficiency of the enzyme coproporphyrinogen oxidase (CPOX). Patients with HCP show <50% of normal activity and those with the rare autosomal recessive harderoporphyria accumulate harderoporphyrinogen, an intermediate porphyrin of the CPOX reaction. To clarify the relationship of the low enzyme activity with these diseases, we expressed mutant CPOX carrying His-tag from these porphyria patients and co-expressed mutant CPOX carrying His-tag and normal CPOX carrying HA-tag in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms a heterodimer in association with the HA-enzyme, and analysis using a cross-link reagent confirmed that the enzyme is a dimer (∼70 kDa). Then, we expressed homo- and heterodimers composed of the wild-type (wt) and engineered mutants of the enzyme or mutants from HCP patients. The monomer form of mutated CPOX did not show any activity and homodimeric enzymes derived from HCP mutant showed low activity (<20% of the control). Some mutations of amino acids 401-404 were associated with marked accumulation of harderoporphyrinogen, with a decrease in the production of protoporphyrinogen, whereas K404E derived from patients with harderoporphyria produced less harderoporphyrinogen. The heterodimers with wt and mutated subunits from HCP patients showed low protoporphyrinogen producing activity. These results show that the substitution of amino acids from R401 to K404 results in extremely low enzyme activity with either mutant homodimer or heterodimers containing normal and mutated subunits and can be linked to HCP disease.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking.

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 µM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Two proteins with different functions are derived from the KlHEM13 gene.

Two proteins that differ at the N terminus (l-KlCpo and s-KlCpo) are derived from KlHEM13, a single-copy-number gene in the haploid genome of Kluyveromyces lactis. Two transcriptional start site (tss) pools are detectable using primer extension, and their selection is heme dependent. One of these tss pools is located 5' of the first translation initiation codon (TIC) in the open reading frame of KlHEM13, while the other is located between the first and second TICs. In terms of functional significance, only s-KlCpo complements the heme deficiency caused by the Δhem13 deletion in K. lactis. Data obtained from immune detection in subcellular fractions, directed mutagenesis, chromatin immunoprecipitation (ChIP) assays, and the functional relevance of ΔKlhem13 deletion for KlHEM13 promoter activity suggest that l-KlCpo regulates KlHEM13 transcription. A hypothetical model of the evolutionary origins and coexistence of these two proteins in K. lactis is discussed.

RLIN1, encoding a putative coproporphyrinogen III oxidase, is involved in lesion initiation in rice.

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen III oxidase in tetrapyrrole biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.

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.

Computational characterization of the substrate-binding mode in coproporphyrinogen III oxidase.

Oxygen-dependent coproporphyrinogen III oxidase catalyzes the sequential decarboxylation of the propionate substituents present on the A and B rings of coproporphyrinogen III in the heme biosynthetic pathway. Although extensive experimental investigation of this enzyme has already afforded many insights into its reaction mechanism, several key features (such as the substrate binding mode, the characterization of the active site, and the initial substrate protonation state) remain poorly described. The molecular dynamics simulations described in this paper enabled the determination of a very promising substrate binding mode and the extensive characterization of the enzyme active site. The proposed binding mode is fully consistent with the known selectivity of the active site toward substituted tetrapyrroles and explains the lack of activity of the H131A, R135A, D274A, and R275A mutants and the reasons behind the nonoccurrence of catalysis on the C and D rings of the tetrapyrrole. An important role in this binding mode is fulfilled by G276, as its carbonyl oxygen intervenes in the substrate anchoring by hydrogen bonding its ring D pyrrole NH group. The presence of this interaction (which is only possible with the protonated NH pyrrole group) and the absence of positively charged side chains close to the pyrrole nitrogen (which might stabilize the N-deprotonated pyrrole postulated in some mechanistic proposals) show that the pyrrole ring is very unlikely to undergo deprotonation during the catalytic cycle and allow the discrimination between the previously postulated mechanistic proposals.

Structure-guided rule-based annotation of protein functional sites in UniProt knowledgebase.

The rapid growth of protein sequence databases has necessitated the development of methods to computationally derive annotation for uncharacterized entries. Most such methods focus on "global" annotation, such as molecular function or biological process. Methods to supply high-accuracy "local" annotation to functional sites based on structural information at the level of individual amino acids are relatively rare. In this chapter we will describe a method we have developed for annotation of functional residues within experimentally-uncharacterized proteins that relies on position-specific site annotation rules (PIR Site Rules) derived from structural and experimental information. These PIR Site Rules are manually defined to allow for conditional propagation of annotation. Each rule specifies a tripartite set of conditions whereby candidates for annotation must pass a whole-protein classification test (that is, have end-to-end match to a whole-protein-based HMM), match a site-specific profile HMM and, finally, match functionally and structurally characterized residues of a template. Positive matches trigger the appropriate annotation for active site residues, binding site residues, modified residues, or other functionally important amino acids. The strict criteria used in this process have rendered high-confidence annotation suitable for UniProtKB/Swiss-Prot features.

Normal and abnormal heme biosynthesis. 6. Synthesis and metabolism of a series of monovinylporphyrinogens related to harderoporphyrinogen. Further insights into the oxidative decarboxylation of porphyrinogen substrates by coproporphyrinogen oxidase.

A series of vinylporphyrinogens were prepared to probe the enzyme coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,c-biladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral substituents, was also a substrate for CPO. Furthermore, a nonnatural type I isomer of harderoporphyrinogen was shown to be acted on by CPO, but in this case further metabolism was noted and this afforded an unprecedented trivinyl porphyrinogen product. The corresponding porphyrin methyl ester was isolated and characterized by FAB MS and proton NMR spectroscopy. The results from these studies allow the binding requirements of CPO to be further assessed and provide a series of substrates to investigate this poorly understood enzyme.

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol.

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite.

Cloning, expression, and biochemical properties of CPOX4, a genetic variant of coproporphyrinogen oxidase that affects susceptibility to mercury toxicity in humans.

Coproporphyrinogen oxidase (CPOX) catalyzes the two-step decarboxylation of coproporphyrinogen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg(2+) present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was twofold lower than that of CPOX (CPOX K(m) = 0.30 microM, V(max) = 0.52 pmol protoporphyrin-IX; CPOX4 K(m) = 0.54 microM, V(max) = 0.33 pmol protoporphyrin-IX). Hg(2+) specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40-50%) than those of either wild-type or heterozygous. Additionally, enzymes from mutant, heterozygous and wild-type livers were comparably inhibited by Hg(2+) (10 microM), decreasing CPOX4 activity to 25% that of the wild-type enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.

Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus.

During haem and chlorophyll biosynthesis, flavin-dependent protoporphyrinogen IX oxidase catalyses the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX. In the following step, iron is inserted into protoporphyrin IX by ferrochelatase. Based on the solved crystal structures of these enzymes, an in silico model for a complex between these two enzymes was proposed to protect the highly photoreactive intermediate protoporphyrin IX. The existence of this complex was verified by two independent techniques. First, co-immunoprecipitation experiments using antibodies directed against recombinantly produced and purified Thermosynechococcus elongatus protoporphyrinogen IX oxidase and ferrochelatase demonstrated their physical interaction. Secondly, protein complex formation was visualized by in vivo immunogold labelling and electron microscopy with T. elongatus cells. Finally, oxygen-dependent coproporphyrinogen III oxidase, which catalyses the formation of protoporphyrinogen IX, was not found to be part of this complex when analysed with the same methodology.

The Radical SAM Superfamily.

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.

A comparative density-functional study of the reaction mechanism of the O2-dependent coproporphyrinogen III oxidase.

During heme biosynthesis, coproporphyrinogen III oxidase catalyzes the conversion of two propionate substituents from the highly reactive substrate coproporphyrinogen III into vinyl substituents, yielding protoporphyrinogen IX. Although the crystal structure of this important enzyme has recently been reported, the reaction mechanism of this intriguing enzyme remains the subject of intense speculation, as impairment of this enzyme has been shown to be the molecular cause behind hereditary coproporphyria. We have performed DFT calculations on model systems in order to analyze several reaction mechanisms proposed for this enzyme. The results afford a full description of the different proposals and allow the rejection of a direct electron abstraction from the protonated substrate by dioxygen. We found that O(2) addition to the (preferentially deprotonated) pyrrole substrate (yielding a hydroperoxide, which then abstracts a proton from the reactive propionate substituent) is compatible with the observed experimental reaction rate, and that the reaction may then proceed through HO2- elimination, followed by decarboxylation.

Use of di- and tripropionate substrate analogs to probe the active site of human recombinant coproporphyrinogen oxidase.

BACKGROUND: Defects in the enzyme coproporphyrinogen oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human coproporphyrinogen oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrinogen-III. These substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. MATERIAL/METHODS: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K(m) and V(max), were determined. RESULTS: Relative to the authentic substrate, the K(m) values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K(m) values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V(max) values were an apparent function of the carbon length of the substituent on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. CONCLUSIONS: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme.

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase.

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.