Nitric oxide and carbon monoxide equilibriums of horse myoglobin and (N-methylimidazole)protoheme. Evidence for steric interaction with the distal residues

Romberg, Robert W.; Kassner, Richard J.

doi:10.1021/bi00591a020

Cited by 61 publications

(45 citation statements)

References 29 publications

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“…Intriguingly, CO-exposed, but not NO-exposed, oxy DosT caused a characteristic change in electronic absorption spectra (SI Fig. 11), which is highly indicative of a heme-carbonyl complex (16,17). In sum, these data conclusively demonstrate that NO and CO are ligands of ferrous DosS and deoxy DosT.…”

Section: Doss Is In the Metmentioning

confidence: 69%

“…The spectral changes observed were highly characteristic of the formation of nitrosylated and carbonylated heme species (Fig. 3C) (16,17). To further confirm that NO is a ligand of DosT, we used EPR to analyze deoxy DosT exposed to proline NONOate.…”

Section: Doss Is In the Metmentioning

confidence: 92%

“…3A). These changes are highly characteristic of heme-NO and heme-CO complexes (16,17). To further validate that NO binds the heme iron of DosS, we used EPR spectroscopy.…”

Section: Doss Is In the Metmentioning

confidence: 99%

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Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Kumar¹,

Toledo

Patel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

300

375

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A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe 3؉ ) form, whereas DosT exists in the O 2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O 2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O 2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O 2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.carbon monoxide ͉ dormancy ͉ nitric oxide ͉ oxygen ͉ persistence T uberculosis (TB) is a major global health burden, and current estimates suggest that one-third of the world's population (Ϸ2 billion) is latently infected with TB (1). Latency is important largely because persistent Mycobacterium tuberculosis (Mtb) are in a state of ''drug unresponsiveness'' wherein the bacilli are resistant to existing antimycobacterial drugs. A major question in the TB field is: ''what are the mechanisms that allow Mtb to persist in human tissues for decades without replicating, to then abruptly resume growth and cause disease?'' Addressing that question is essential to the development of effective therapeutic intervention strategies. Recent evidence implicates NO as an environmental trigger of mycobacterial persistence (2-5). The latter findings are particularly interesting in light of the fact that inducible NO synthase (iNOS) and therefore NO production is crucial for protection of mice against Mtb (6), and that human macrophages in Mtb-infected tissues express iNOS (2, 7). Another factor associated with latent TB is hypoxia (8). The role of oxygen tension in TB is receiving wide attention, especially because it was demonstrated that rapid withdrawal of oxygen is lethal to Mtb, whereas a gradual depletion allows time for adaptation and bacterial survival (8). Interestingly, a significant overlap exists between the gene expression profiles of Mtb cells treated with NO and that of bacilli cultured under hypoxic conditions (3-5). ...

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Section: Doss Is In the Metmentioning

confidence: 69%

Section: Doss Is In the Metmentioning

confidence: 92%

See 1 more Smart Citation

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Kumar¹,

Toledo

Patel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

300

375

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“…The behavior ofa model having an anthracene suspended above a porphyrin has given support to the conclusion that steric effects can weaken CO binding to heme iron (13). Studies on the equilibrium constants for CO and NO binding, both to model compounds and to heme proteins, confirm the role of a steric effect in weakening the binding of CO to myoglobin (19).…”

Section: Structural Analyses Of Carbonyl Hemoglobins and Carbonyl Myomentioning

confidence: 89%

Steric control of CO binding in a totally synthetic heme protein model

Busch

Zimmer

Grzybowski

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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A family oftotally synthetic models for the carbon Rt monoxide adducts of heme proteins has been synthesized and ap-R2/ plied to the elucidation of the role of steric (13,14). It has been proposed that the crowding is responsible for the partial detoxification ofcarbon monoxide in respiring organisms (4, 11). For example, the ratio of equilibrium constants (12-18) for CO and 02 binding (formation) Kco/Ko2 for a protein-free modified heme having no structural counterpart to the distal imidazole (Traylor's chelated protoheme) is 840, whereas the corresponding ratio in myoglobin under comparable conditions is 20 (15). The discrimination against carbon monoxide by myoglobin is evident from the fact that the equilibrium constant for 02 binding for the chelated protoheme (2.0 x 106 M-1) is very close to the value for myoglobin (1.5 x 106 M-1). The behavior ofa model having an anthracene suspended above a porphyrin has given support to the conclusion that steric effects can weaken CO binding to heme iron (13). Studies on the equilibrium constants for CO and NO binding, both to model compounds and to heme proteins, confirm the role of a steric effect in weakening the binding of CO to myoglobin (19). In contrast, there is no substantial selective process, steric or otherwise, operating against the binding to iron of CO, as compared to 02, in the case of Hb(R) (12). These results generate two related questions, because the FeCO linkage is distorted (1-3) in both crystalline Hb(R)(CO) and crystalline Mb(CO): (i) Do steric effects that push the normally linear FeCO group into some off-axis configuration necessarily decrease the CO affinities of iron(II) complexes? (ii) If yes, are there structural differences between the carbon monoxide complex of Hb(R)(CO) in aqueous solutions or suspension (20) and in its crystalline state that would account for the apparent contradiction?The structural origins of modifications in 02 affinity are well illustrated by the T and R forms ofhemoglobin. For 02 binding, the equilibrium constant is altered by changes in the rate of dissociation and is attributable to constraints placed on the proximal imidazole by protein conformation and the ease with which the iron atom may occupy the N4 plane of the protoporphyrin (4-11, 21).We report here a systematic and definitive demonstration that steric effects can indeed control the CO affinity of iron(II). This is shown by direct equilibrium measurements with a family ofnonporphyrin heme protein models coupled with key crystal structure analyses. The ligands in our models contain superstructure components that facilitate control of the space available for the binding of CO to an iron(II) atom (22). These socalled lacunar ligands are neutral, bicyclic molecules having conformations that incorporate permanent voids in the vicinity of a metal coordination site (see Fig. 1). The efficacy of these synthetic species as biological mimics has been shown through studies on the reversibly formed 1:1 oxygen adducts of the cobalt(II) and iron(II) compl...

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“…1B and Supplementary Table S1). These spectral changes are characteristic of the nitrosyl heme complex (21,25), suggesting that NO forms a nitrosyl heme complex with SenX3. We also exposed SenX3 to an equal amount of spent ProliNONOate.…”

Section: Senx3 Interacts With O 2 No and Comentioning

confidence: 92%

Virulence Factor SenX3 Is the Oxygen-Controlled Replication Switch ofMycobacterium tuberculosis

Singh

Kumar²

2015

Antioxidants & Redox Signaling

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Aim: Morphogenetic switching between the replicating and nonreplicating states of Mycobacterium tuberculosis is regulated by oxygen, nitric oxide, and carbon monoxide levels. The mechanisms by which M. tuberculosis senses these diatomic gases remain poorly understood. In this study, we have examined whether virulence factor SenX3 plays any role in oxygen sensing. Results: In this study, we demonstrate that the virulence factor SenX3 is a heme protein that acts as a three-way sensor with three levels of activity. The oxidation of SenX3 heme by oxygen leads to the activation of its kinase activity, whereas the deoxy-ferrous state confers a moderate kinase activity. The binding of nitric oxide and carbon monoxide inhibits kinase activity. Consistent with these biochemical properties, the SenX3 mutant of M. tuberculosis is capable of attaining a nonreplicating persistent state in response to hypoxic stress, but its regrowth on the restoration of ambient oxygen levels is significantly attenuated compared with the wild-type and the complemented mutant strains. Furthermore, the presence of signaling concentrations of nitric oxide and carbon monoxide was able to inhibit the regrowth of M. tuberculosis in response to ambient oxygen levels. Innovation and Conclusions: Evidence presented in this study delineates a plausible mechanism explaining the oxygen-induced reactivation of tuberculosis diseases in humans after many years of latent infection. Furthermore, this study implicates nitric oxide and carbon monoxide in the inhibition of mycobacterial growth from the nonreplicating state. Antioxid. Redox Signal. 22,[603][604][605][606][607][608][609][610][611][612][613]

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Nitric oxide and carbon monoxide equilibriums of horse myoglobin and (N-methylimidazole)protoheme. Evidence for steric interaction with the distal residues

Cited by 61 publications

References 29 publications

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Steric control of CO binding in a totally synthetic heme protein model

Virulence Factor SenX3 Is the Oxygen-Controlled Replication Switch ofMycobacterium tuberculosis

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