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.

Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (•NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of •NO with O ₂ , reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of •NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 μM) O ₂ concentrations and exhibits zero-order dependence on •NO concentration. Indeed, RSNO formation is not inhibited even at O ₂ levels 50%. One possible metal-dependent, O ₂ -independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of •NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by ≈50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free •NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells.

One of the most important biological reactions of nitric oxide (nitrogen monoxide, •NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g = 2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous •NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, •NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from •NO, and SIH added during the •NO treatment “freezes” DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free •NO, because addition of oxyhemoglobin prevents formation from either •NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by •NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free •NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of •NO and its effects on cellular iron homeostasis.