IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Loutet, Slade A.; Kobylarz, M.J.; Chau, Crystal H.T.; Murphy, M.E.P.

doi:10.1074/jbc.m113.470518

Cited by 24 publications

(40 citation statements)

References 83 publications

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“…In studies of both canonical and IsdG-type heme oxygenases, the electrons have been supplied by excess ascorbate, dithionite, or other chemical donors that can act as surrogates for enzymatic reductases proposed to act in concert with the oxygenases in the cell. 13, 20, 24, 53, 54 Under conditions that led to reactivity in IsdG-type heme oxygenases (excess ascorbate, O 2 and catalase to consume H 2 O 2 ), 13, 20 no reaction was observed for the decarboxylase-coproheme complex (i.e., no changes in the UV/vis spectrum and no loss/conversion of coproheme was observed by HPLC, Figure S2). Reactions were subsequently carried out starting from the ferrous coproheme-decarboxylase complex.…”

Section: Resultsmentioning

confidence: 99%

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

et al. 2016

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“…Since IsdG enzymes have been discovered in several human pathogens, including S. aureus , 5 Bacillus anthracis , 37 and Listeria monocytogenes , 38 they have been identified as potential antibiotic targets. 22, 39, 40 The first inhibitors of IsdG identified were non-iron metalloporphyrins (Fig. 8), 32 but the tight binding of ferric heme by IsdG now seems to rule out competitive inhibition as a viable strategy.…”

Section: Discussionmentioning

confidence: 99%

“…A potential explanation for these observations relates to the fact that the K d values for IsdG–heme and IsdI–heme were determined by fitting UV/Vis absorption titration data to a Michaelis-Menten model. 5 Subsequent research has called into question whether this enzyme follows Michaelis-Menten kinetics due to the requirement of at least three substrates, 5, 7, 11 plus a reductase, 22 and the presence of at least two enzymatic intermediates. 11 Thus, the dissociation constants for IsdG–heme and IsdI–heme were re-investigated.…”

Section: Introductionmentioning

confidence: 99%

Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor

2017

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The micromolar equilibrium constants for heme dissociation from IsdG and IsdI reported in the literature call into question whether these enzymes are actually members of the iron-regulated surface determinant system of Staphylococcus aureus, which harvests heme iron from a host during infection. In order to address this question, the heme dissociation constants for IsdG and IsdI were reevaluated using three approaches. The heme dissociation equilibrium constants were measured using a UV/Vis absorption-detected assay analyzed with an assumption-free model, and using a newly developed fluorescence-detected assay. The heme dissociation rate constants were estimated using apomyoglobin competition assays. Analyses of the UV/Vis absorption data revealed a critical flaw in the previous measurements; heme is 99.9% protein-bound at the micromolar concentrations needed for UV/Vis absorption spectroscopy, which renders accurate equilibrium constant measurement nearly impossible. However, fluorescence can be measured for more dilute samples, and analyses of these data resulted in dissociation equilibrium constants of 1.4 ± 0.6 nM and 12.9 ± 1.3 nM for IsdG and IsdI, respectively. Analyses of the kinetic data obtained from apomyoglobin competition assays estimated heme dissociation rate constants of 0.022 ± 0.002 s-1 for IsdG and 0.092 ± 0.008 s-1 for IsdI. Based upon these data, and what is known regarding the post-translational regulation of IsdG and IsdI, it is proposed that only IsdG is a member of the heme iron acquisition pathway and IsdI regulates heme homeostasis. Furthermore, the nanomolar dissociation constants mean that heme is bound tightly by IsdG and indicates that competitive inhibition of this protein will be difficult. Instead, uncompetitive inhibition based upon a detailed understanding of enzyme mechanism is a more promising antibiotic development strategy.

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“…Both enzymes degrade heme to a mixture of β-and δ-staphylobilin in the presence of oxygen and the reductase IruO [11][12][13], although the ratio of the two isomeric products produced by each enzyme is significantly different. As is the case for canonical heme oxygenase-1 and heme oxygenase-2 [14], IsdG and IsdI have distinct post-translational regulation.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure

et al. 2015

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Staphylococcus aureus IsdG catalyzes the final step of staphylococcal iron acquisition from host hemoglobin, whereby host-derived heme is converted to iron and organic products. The Asn7 distal pocket residue is known to be critical for enzyme activity, but the influence of this residue on the substrate electronic structure was unknown prior to this work. Here, an optical spectroscopic and density functional theory characterization of azide- and cyanide-inhibited wild type and N7A IsdG is presented. Magnetic circular dichroism data demonstrate that Asn7 perturbs the electronic structure of azide-inhibited, but not cyanide-inhibited, IsdG. As the iron-ligating α-atom of azide, but not cyanide, can act as a hydrogen bond acceptor, these data indicate that the terminal amide of Asn7 is a hydrogen bond donor to the α-atom of a distal ligand to heme in IsdG. Circular dichroism characterization of azide- and cyanide-inhibited forms of WT and N7A IsdG strongly suggests that the Asn7···N3 hydrogen bond influences the orientation of a distal azide ligand with respect to the heme substrate. Specifically, density functional theory calculations suggest that Asn7···N3 hydrogen bond donation causes the azide ligand to rotate about an axis perpendicular to the porphyrin plane and weakens the π-donor strength of the azide ligand. This lowers the energies of the Fe 3d xz and 3d yz orbitals, mixes Fe 3d xy and porphyrin a 2u character into the singly-occupied molecular orbital, and results in spin delocalization onto the heme meso carbons. These discoveries have important implications for the mechanism of heme oxygenation catalyzed by IsdG.

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IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Cited by 24 publications

References 83 publications

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor

Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure

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IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Cited by 24 publications

References 83 publications

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b

Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor

Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure

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Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O₂ and H₂O₂ Yield Ferric Heme b