New Insights Into the Function of Flavohemoglobin in <i>Mycobacterium tuberculosis</i>: Role as a NADPH-Dependent Disulfide Reductase and D-Lactate-Dependent Mycothione Reductase

Mycobacterium tuberculosis ( Mtb ) produces an unconventional flavohemoglobin ( Mtb FHb) that carries a FAD-binding site similar to D-lactate dehydrogenases (D-LDH) and oxidizes D-lactate into pyruvate. The molecular mechanism by which Mtb FHb functions in Mtb remains unknown. We discovered that the D-LDH-type FAD-binding site in Mtb FHb overlaps with another FAD-binding motif similar to thioredoxin reductases and reduces DTNB in the presence of NADPH similar to trxB of Mtb . These results suggested that Mtb FHb is functioning as a disulfide oxidoreductase. Interestingly, D-lactate created a conformational change in Mtb FHb and attenuated its ability to oxidize NADPH. Mass spectroscopy demonstrated that Mtb FHb reduces des- myo- inositol mycothiol in the presence of D-lactate unlike NADPH, indicating that D-lactate changes the specificity of Mtb FHb from di-thiol to di-mycothiol. When M. smegmatis carrying deletion in the fhb II gene (encoding a homolog of Mtb FHb) was complemented with the fhb gene of Mtb , it exhibited four- to fivefold reductions in lipid peroxidation and significant enhancement in the cell survival under oxidative stress. These results were corroborated by reduced lipid peroxidation and enhanced cell survival of wild-type M. smegmatis after overexpression of the fhb gene of Mtb . Since D-lactate is a by-product of lipid peroxidation and Mtb FHb is a membrane-associated protein, D-lactate-mediated reduction of mycothiol disulfide by Mtb FHb may uniquely equip Mtb to relieve the toxicity of D-lactate accumulation and protect the cell from oxidative damage, simultaneously balancing the redox environment under oxidative stress that may be vital for the pathogenesis of Mtb .