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

Abstract

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...

Molecular hijacking of siroheme for the synthesis of heme and d 1 heme

“…Overall, this process is similar to the HemN catalyzed oxidative decarboxylation reaction of coproporphyrinogen III to protoporphyrinogen IX that occurs during the classical heme biosynthesis pathway (27,28). As with HemN (and AhbC), AhbD is also a radical AdoMet family member.…”

“…Recombinant Rhodobacter sphaeroides 5-aminolevulinic acid synthase (HemA) was produced and purified as described in ref. 28. Recombinant E. coli succinyl-coenzymeA synthetase (SucCD) was purified as a hexahistidine fusion protein as follows.…”

Nitrous Oxide Reduction by an Obligate Aerobic Bacterium, Gemmatimonas aurantiaca Strain T-27

“…The oxygen dependence of C. jejuni strain 11168 was attributed to the requirement for oxygen-dependent ribonucleotide reductase (RNR). Another plausible explanation for the oxygen requirement of strictly aerobic bacteria is the use of HemF, the oxygen-dependent coproporphyrinogen III oxidase, for coproporphyrinogen III decarboxylation in heme synthesis (46). Neither case applies to G. aurantiaca strain T-27, as it possesses the genes encoding class II RNR and HemN, the oxygenindependent counterparts for class I RNR and HemF, respectively, in its genome; however, it is still possible that other essential metabolic functions for biosynthesis in G. aurantiaca strain T-27 may be carried out by oxygen-dependent enzymes.…”

Discovery of a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through Comparative Genomic Analysis and Functional Complementation

“…The first gap occurs during the cyclization of the linear tetrapyrrole, where uroporphyrinogen synthase cannot be identified in some Alphaproteobacteria and Chlamydiaceae. The next missing step is the antepenultimate step, where two distinct enzymes are known to catalyze this reaction (7), i.e., an oxygen-dependent coproporphyrinogen oxidase named HemF (3) and an oxygen-independent, radical S-adenosyl-L-methionineutilizing coproporphyrinogen dehydrogenase named HemN (29). Some heme-synthesizing bacteria lack both of these proteins, so it is clear that there is an as-yet-undiscovered form of coproporphyrinogen oxidase.…”