A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ) the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though significantly greater than the expected two equivalents were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high valent iron intermediates cannot be ruled out.

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr■. This residue then abstracts a net hydrogen atom (H■) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e−/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Chlorite dismutases (Clds) convert chlorite to O2 and Cl–, stabilizing heme in the presence of strong oxidants and forming the O=O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

Heme oxygenases (HOs) 3 are enzymes that oxidatively liberate iron from the heme tetrapyrrole (1-3). In the well characterized HOs from animals and many bacteria, the same heme molecule acts as both the O 2 -activating cofactor and substrate. Three successive monooxygenation steps yield Fe(II), CO, and biliverdin IX␣ as the end products of the reaction (Fig. 1) (2, 3). Animals use HOs to maintain cellular heme homeostasis as part of a constant cycle of heme synthesis and breakdown. The products report on the status of this cycle and serve as antioxidants and signaling agents (4, 5). Many bacteria also use HO homologs, both to control heme homeostasis and to liberate iron from host-derived heme (6, 7). Heme, found primarily in hemoglobin, can therefore be used as a rich nutritional source of iron. Because of the intriguing nature of the reaction, which uses heme as both cofactor and substrate (8 -10), as well as the acute biological importance of HO-mediated processes, HOs from several species have been exceptionally well characterized (2, 3).By the early 2000s, however, it was apparent that many important Gram-positive pathogens that degrade host heme did not possess an HO-encoding gene in their genomes. A new family of heme-degrading proteins known as IsdGs was subsequently discovered, with representatives found in bacteria from both Gram-positive and Gram-negative phyla (11). IsdG family proteins are evolutionarily and structurally distinct from the well studied HOs (12, 13), and they yield different end products. Instead of biliverdin IX␣ and CO, the IsdG protein from Mycobacterium tuberculosis (known as MhuD) generates triply-oxygenated linear tetrapyrroles called mycobilins (Fig. 1) (14, 15). A formyl group remains appended to pyrrole ring A or B at the site of macrocycle cleavage, and an oxo group is generated on the pyrrole ring on the opposite side. Notably, no C1 product is released (16).Although homologous to MhuD, the IsdG from Staphylococcus aureus degrades heme to yet a third set of products. The macrocycle is not cleaved at the ␣-meso-but rather at either the ␤-or ␦-meso-carbon. Oxo groups are generated on both the carbon backbone and the pyrrole rings at the cleavage site, generating tetrapyrrole products known as staphylobilins (Fig. 1) (17). It was recently shown that a C1 product is indeed released by the S. aureus IsdG; however, quite unexpectedly, the major C1 product was determined to be formaldehyde (CH 2 O) instead of CO (18). Unlike CO, formaldehyde may be undetectable by animal immune systems, offering a potential selective advantage for heme-feeding pathogens that use IsdG-type enzymes (5,19,20). Mechanistically, the observation that CH 2 O instead of CO implies that verdoheme, the green inter-* This work was supported in part by National Institutes of Health Grants GM090260 and 5P20RR02437 of the CoBRE Program (to J. L. D.) and Grant RO1 AI069233 (to E. P. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the resp...

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PFam Clan 0032, also known as the CDE superfamily, is a diverse group of at least 20 protein families sharing a common α, β-barrel domain. Of these, six different groups bind heme inside the barrel’s interior, using it alternately as a cofactor, substrate, or product. Focusing on these six, an integrated picture of structure, sequence, taxonomy, and mechanism is presented here, detailing how a single structural motif might be able to mediate such an array of functions with one of nature’s most important small molecules.

Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH dependent, and further decelerated by D2O2 (average KIE = 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme-proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.