Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo 3 (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.anabolism coupled catabolism | protoporphyrinogen IX oxidase | HemG | tetrapyrrole | respiration

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 ␤-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 15 N) 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 ␣-and ␤-positions, or only at the ␤-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the ␣-, ␤-, and methylene bridge positions. Deuterium or 15 N 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-Shydrogen atom at the ␤-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.Modified tetrapyrroles such as hemes and chlorophylls play important roles in a range of essential life processes from respiration to photosynthesis. Their underlying molecular architecture is reflected in a shared biosynthetic pathway that requires the coordinated activity of a large number of highly diverse enzymes (1-3). During porphyrin formation two structurally unrelated coproporphyrinogen III oxidases catalyze the oxidative decarboxylation of the propionate side chains on pyrrole rings A and B of the macrocycle to the corresponding vinyl groups (Scheme 1a) (4). The oxygen-dependent enzyme, HemF, found in eukaryotes and some bacteria, uses molecular oxygen as an electron acceptor during this process (5). For oxygen-independent coproporphyrinogen formation most bacteria carry the oxygen-independent enzyme HemN. In Escherichia coli, HemN is a monomeric protein that contains an oxygen-sensitive [4Fe-4S] cluster (6, 7). The enzyme belo...

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygendependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a K m value of 2.6 M for coproporphyrinogen III with a k cat value of 0.17 min ؊1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the en-

The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified

A4 Structural diversity in metal ion chelation, and the structure of uro'gen 111 synthasc

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During the biosynthesis of hemes and chlorophylls coproporphyrinogen 111 oxidase decarboxylates coproporphyrinogen I11 to form protoporpyhrinogen IX. In eucaryotes almost exclusively an oxygen-dependent enzyme is found. Due to anaerobic growth environments bacteria possess two structurally not related enzymes, again the oxygen-dependent HemF and the oxygen-independent HemN. Both enzymes from Escherichia coli were biochemically characterized after recombinant production and chromatographic purification. Oxygen-dependent HemF requires histidine coordinated manganese. Oxygen-independent H e m N carrying an iron-sulphur-cluster requires S-adenosyl methionine, NAD(P)H and an unknown cytoplasmic component for activity. Similarities with enzymes using radical mechanisms are obvious.16 Mechanistic insights into flavocytochrome c3, the soluble fumarate reductase from Shewanella frigidimarina.