The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H2O2, other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys60 as the "peroxidatic" cysteine (CP) forming a complex catalytic center with Cys93 as the "resolving" cysteine (CR). Site-directed mutagenesis confirms Cys60 to be CP and Cys80 to be catalytically irrelevant. Replacing Cys93 with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys60 to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys60 and Cys93 and between Cys60 and Cys80 in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys60 and never to Cys93 of the TPx. It is concluded that neither Cys80 nor Cys93 is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys60 being the site of attack for both the oxidizing and the reducing substrate. The role of Cys93 is likely to conserve the oxidation equivalents of the sulfenic acid state of CP as a disulfide bond to prevent overoxidation of Cys60 under a restricted supply of reducing substrate.