Porphyrin Biosynthesis Research Articles

Computational studies on Schiff-base formation: Implications for the catalytic mechanism of porphobilinogen synthase

Schiff bases are common and important intermediates in many bioenzymatic systems. The mechanism by which they are formed, however, is dependent on the solvent, pH and other factors. In the present study we have used density functional theory methods in combination with appropriate chemical models to get a better understanding of the inherent chemistry of the formation of two Schiff bases that have been proposed to be involved in the catalytic mechanism of porphobilinogen synthase (PBGS), a key enzyme in the biosynthesis of porphyrins. More specifically, we have investigated the uncatalysed reaction of its substrate 5-aminolevulinic acid (5-ALA) with a lysine residue for the formation of the P-site Schiff base, and as possibly catalysed by the second active site lysine, water or the 5-ALA itself. It is found that cooperatively both the second lysine and the amino group of the initial 5-ALA itself are capable of reducing the rate-limiting energy barrier to 14.0 kcal mol −1. We therefore propose these to be likely routes involved in the P-site Schiff-base formation in PBGS.

The first branching point in porphyrin biosynthesis: A systematic docking, molecular dynamics and quantum mechanical/molecular mechanical study of substrate binding and mechanism of uroporphyrinogen-III decarboxylase

In humans, uroporphyrinogen decarboxylase is intimately involved in the synthesis of heme, where the decarboxylation of the uroporphyrinogen-III occurs in a single catalytic site. Several variants of the mechanistic proposal exist; however, the exact mechanism is still debated. Thus, using an ONIOM quantum mechanical/molecular mechanical approach, the mechanism by which uroporphyrinogen decarboxylase decarboxylates ring D of uroporphyrinogen-III has been investigated. From the study performed, it was found that both Arg37 and Arg50 are essential in the decarboxylation of ring D, where experimentally both have been shown to be critical to the catalytic behavior of the enzyme. Overall, the reaction was found to have a barrier of 10.3 kcal mol(-1) at 298.15 K. The rate-limiting step was found to be the initial proton transfer from Arg37 to the substrate before the decarboxylation. In addition, it has been found that several key interactions exist between the substrate carboxylate groups and backbone amides of various active site residues as well as several other functional groups.

ChemInform Abstract: The Synthesis of a Fluorinated Analogue of 5-Aminolaevulinic Acid, a Potential Inhibitor of Porphyrin Biosynthesis.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Photosensitive Rash in Association with Porphyrin Biosynthesis Possibly Induced by Docetaxel and Trastuzumab Therapy in a Patient with Metastatic Breast Carcinoma

Docetaxel (Taxotere), an anticancer agent, is known to cause various reactions, including hypersensitivity, oedema, skin toxicity with erythrodysesthesia syndrome, infusion site reactions, alopecia, nail onycholysis, nail pigmentation, photosensitivity, scleroderma and stomatitis. However, of all the reported effects, photosensitivity has only rarely been described in the literature. We experienced a case of cutaneous photosensitivity with aberrations in porphyrin biosynthesis that developed 1 month after the patient received combination chemotherapy consisting of docetaxel and trastuzumab. The eruption resolved with sun avoidance and discontinuation of docetaxel therapy. To our knowledge, this is the first case of a photosensitive reaction with enhanced levels of porphyrins during docetaxel therapy.

Targeting the Active Site Gate to Yield Hyperactive Variants of 5-Aminolevulinate Synthase

The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5'-phosphate-dependent enzyme 5-aminolevulinate synthase (EC 2.3.1.37). Based on the postulate that turnover in this enzyme is controlled by conformational dynamics associated with a highly conserved active site loop, we constructed a variant library by targeting imperfectly conserved noncatalytic loop residues and examined the effects on product and porphyrin production. Functional loop variants of the enzyme were isolated via genetic complementation in Escherichia coli strain HU227. Colony porphyrin fluorescence varied widely when bacterial cells harboring the loop variants were grown on inductive media; this facilitated identification of clones encoding unusually active enzyme variants. Nine loop variants leading to high in vivo porphyrin production were purified and characterized kinetically. Steady state catalytic efficiencies for the two substrates were increased by up to 100-fold. Presteady state single turnover reaction data indicated that the second step of quinonoid intermediate decay, previously assigned as reaction rate-limiting, was specifically accelerated such that in three of the variants this step was no longer kinetically significant. Overall, our data support the postulate that the active site loop controls the rate of product and porphyrin production in vivo and suggest the possibility of an as yet undiscovered means of allosteric regulation.

Exogenous application of 5-aminolevulinic acid increases the transcript levels of sulfur transport and assimilatory genes, sulfate uptake, and cysteine and glutathione contents in Arabidopsis thaliana

5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. Although ALA is known to promote carbon fixation and nitrogen assimilation in plants, the effects of ALA on sulfur assimilation have not been determined. In the present study, we analyzed the effect of ALA on sulfur assimilation. We used a fusion gene construct consisting of a promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis and green fluorescent protein ([GFP] P SULTR1;2 -GFP) to determine whether ALA treatment influences the expression of the sulfur transport gene. The GFP levels in P SULTR1;2 -GFP plants were significantly increased by 0.3 and 1 mmol L−1 ALA under both sulfur-sufficient and sulfur-deficient conditions. Real-time reverse transcription-polymerase chain reaction experiments revealed that these concentrations of ALA also increased the mRNA levels of other key sulfur transport and assimilatory genes, such as SULTR, adenosine 5′-phosphosulfate reductases and serine acetyl transferase. Sulfate uptake was enhanced by ALA treatment under sulfur-sufficient conditions. In addition, ALA treatment increased the accumulation of cysteine and glutathione, particularly in the shoot. Our data demonstrated that exogenously applied ALA increases the transcript levels of some sulfur assimilatory genes, sulfate uptake, and the contents of cysteine and glutathione. We propose a new role for ALA in regulating the sulfur assimilatory pathway.

GUN4 Is Required for Posttranslational Control of Plant Tetrapyrrole Biosynthesis

In aerobic photosynthetic organisms, GUN4 binds the chlorophyll intermediates protoporphyrin and Mg protoporphyrin, stimulates Mg chelatase activity, and is implicated in plastidic retrograde signaling. GUN4 expression is most abundant in young and greening tissues and parallels the activity of 5-aminolevulinic acid (ALA) ALA and Mg porphyrin biosynthesis during photoperiodic growth. We explored function and mode of action of GUN4 using GUN4-deficient and overexpressing plants. GUN4 overexpression leads to a general activation of the enzymes of chlorophyll biosynthesis. During photoperiodic growth GUN4 deficiency prevents ALA synthesis and chlorophyll accumulation. All these metabolic changes do not correlate with altered gene expression or changes of protein abundance in tetrapyrrole biosynthesis. While ALA feeding fails to compensate GUN4 deficiency during light-dark growth, this approach results in chlorophyll accumulation under continuous dim light. A new model defines the involvement of GUN4 in posttranslational regulation of ALA and Mg porphyrin synthesis, to sustain chlorophyll synthesis, namely under varying environmental conditions.

Cell Survival under Stress Is Enhanced by a Mitochondrial ATP-Binding Cassette Transporter That Regulates Hemoproteins

The ATP-binding cassette (ABC) transporter ABCB6 localizes to the mitochondria, where it imports porphyrins and up-regulates de novo porphyrin synthesis. If ABCB6 also increases the intracellular heme concentration, it may broadly affect the regulation and physiology of cellular hemoproteins. We tested whether the ability of ABCB6 to accelerate de novo porphyrin biosynthesis alters mitochondrial and extramitochondrial heme levels. ABCB6 overexpression increased the quantity of cytosolic heme but did not affect mitochondrial heme levels. We then tested whether the increased extramitochondrial heme would increase the concentration and/or activity of cellular hemoproteins (hemoglobin, catalase, and cytochrome c oxidase). ABCB6 overexpression increased the activity and quantity of hemoproteins found in several subcellular compartments, and reduction of ABCB6 function (by small interfering RNA or knockout) reversed these findings. In complementary studies, suppression of ABCB6 expression sensitized cells to stress induced by peroxide and cyanide, whereas overexpression of ABCB6 protected against both stressors. Our findings show that the ability of ABCB6 to increase cytosolic heme levels produces phenotypic changes in hemoproteins that protect cells from certain stresses. Collectively, these findings have implications for the health and survival of both normal and abnormal cells, which rely on heme for multiple cellular processes.

La photothérapie dynamique topique

Topical photodynamic therapy is the destruction of skin lesions by exposure to light after prior application of a precursor in porphyrin biosynthesis: delta-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL). Metvixia® (MAL) was recently authorized in France for the topical photodynamic therapy of actinic keratosis, superficial basal cell carcinoma (except for lesions on the face) and cutaneous squamous cell carcinoma in situ. But other indications for the use of this procedure are beginning to emerge, including other cancers (like extramammary Paget’s disease), infective and inflammatory dermatoses. Photodynamic therapy can be an effective treatment for multiple lesions with an excellent cosmetic result. In conclusion, this technique represents an interesting alternative therapy and could have a place in the management of proctological disease.

Metal Ion Substrate Inhibition of Ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

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.

Melatonin and Environmental Lighting Regulate ALA‐S Gene Expression and So Porphyrin Biosynthesis in the Rat Harderian Gland

The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day‐night PPIX levels as well as day‐night activity and mode of expression of the porphyrinogenic enzymes δ‐aminolevulinate synthase (ALA‐S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630‐ to 670‐fold higher than in the liver, without a day‐night difference, which is the consequence of both low Fech gene expression (5‐ to 7‐fold lower than in the liver) and ALA‐S over‐expression (4‐ to 7‐fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day‐night variation, whereas HG ALA‐S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA‐S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day‐night ALA‐S variation in the HG that is observed under standard 12 L∶12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA‐S gene expression in the rat HG.

Genomic Differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, Identified by Suppression Subtractive Hybridization

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

The ins and outs of tRNA transport

Transfer RNAs (tRNAs) are involved in many cellular functions distributed throughout the cellular space. In addition to their role in protein translation, these molecules are engaged in tasks as diverse as the regulation of gene expression, amino‐acid synthesis, protein degradation, cell‐wall synthesis, porphyrin biosynthesis, priming of replication, RNA interference and the transport of macromolecules. The nucleus and the mitochondria of eukaryotic cells, as well as the chloroplasts in plants, contain their own genome that encodes a range of proteins and nucleic acids. Early evidence suggested that some of the RNA content of mitochondria could be transcribed from non‐mitochondrial DNA (Suyama, 1967). It is now well established that a variable number of tRNA species present in the mitochondria are indeed nucleus‐encoded (Fig 1). The phenomenon of mitochondrial tRNA import has been reported in plants, marsupials, the yeast Saccharomyces cerevisiae , and the protozoa Leishmania , Trypanosoma and Tetrahymena . When certain tRNA species are not encoded by the mitochondrial genome, there is a predictable requirement for the import of nucleus‐encoded tRNAs into the mitochondria (Schneider & Marechal‐Drouard, 2000). This is the case, for example, for the mitochondria of Leishmania , which are completely devoid of tRNA‐encoding genes. However, there are a few examples of tRNA import into mitochondria that already have a complete set of mitochondria‐encoded tRNAs. Other tRNA transport systems have also been identified (Fig 1), which include their retrograde transport from the cytoplasm to the nucleus (Takano et al , 2005; Shaheen & Hopper, 2005), the export of mitochondria‐encoded tRNA to the cytoplasm (Maniataki & Mourelatos, 2005), and the packaging of tRNAs into retroviruses (Waters & Mullin, 1977). The roles of some of these transport systems have yet to be defined. Figure 1. The travels of transfer RNA. …

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.

Identification of a mammalian mitochondrial porphyrin transporter

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

5-Aminolaevulinic acid (ALA) induced formation of different fluorescent porphyrins: A study of the biosynthesis of porphyrins by bacteria of the human digestive tract

Aminolaevulinic acid (ALA) induces porphyrin formation in almost all living cells. The fluorescence spectra of porphyrins produced from a variety of 31 bacterial strains from the human oral cavity and other parts of digestive tract have been examined. Many of the bacteria exposed to ALA were able to induce protoporphyrin IX (PpIX) fluorescence, but under aerobic condition some bacteria can also produced different fluorescent porphyrins, in particular water-soluble porphyrins that can arise from an oxidation of the corresponding porphyrinogen precursors. The formation of fluorescent porphyrins can be different from one bacterial strain to another, but also one specific bacterium can form different fluorescent porphyrins. Irradiation of the ALA incubated cultures led to a rapid formation of water-soluble porphyrins exhibiting fluorescence maxima at wavelengths of 618–620 nm. This light induced formation of water-soluble porphyrins could be attributed to a photooxidation of the non-fluorescent (Uro/Copro)-porphyrinogen precursors. Addition of detergents to some of the bacterial cultures led to a strong PpIX fluorescence increase, indicating that some of the PpIX originally produced can be present in a non-fluorescent, probably aggregated, form. The large abundance of bacteria in the oral cavity and other parts of digestive tract, with their capacity to easily produce fluorescent porphyrins, indicates that such bacterial fluorescence should be suppressed during the ALA-based diagnosis of tumours in order to eliminate false positive results.

The effect of folic acid on porphyrin synthesis in tumors and normal skin of mice treated with 5-aminolevulinic acid or methyl 5-aminolevulinate

5-Aminolevulinic acid (ALA) or its derivative methyl 5-aminolevulinate (MAL) combined with folic acid was applied in nude mice bearing human colon adenocarcinoma. The aim of the study is to see whether folic acid may increase biosynthesis of porphyrins in tumor tissue after systemic or topical administration of ALA or MAL. The production of porphyrins was determined by spectrofluorometric measurements with an optical fibre probe. It was found that the porphyrin production after i.p injection of 200 mg kg(-1) ALA or MAL was significantly increased by i.p injection of 100 mg kg(-1) folic acid. However, in the case of topically applied 20% ALA, folic acid had no effect. In the case of topically applied 20% MAL, folic acid (i.p or topically applied) reduced the porphyrin synthesis. This might be used for the protection of normal skin against photosensitization. The effects of folic acid were similar in tumors and normal skin. Two mechanisms may explain the results: enhancement of the efficiency of the rate-limiting enzyme porphobilinogen deaminase by folic acid or interference of folic acid with the transport of ALA and MAL to and into the cells synthesizing porphyrins in the tissues. The present data seem to favour the latter mechanism. Folic acid may have a role as an adjuvant in photodynamic therapy with systemically administered ALA and its derivatives.

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

The chloroplast outer envelope protein OEP37 is a member of the growing beta-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (lambda) of Lambda= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (P(K+)/P(K-) = 14:1) with a voltage-dependent open probability maximal at V(mem) = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

The Substrate Radical of Escherichia coli Oxygen-independent Coproporphyrinogen III Oxidase HemN

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.