Objective:To investigate the mechanism of Mycobacterium tuberculosis ( Mtb) WhiB2 in the pathogenesis of tuberculosis. Methods:A recombinant vector of pET28a-WhiB2 for heterogeneous expression of WhiB2 was constructed. The target protein WhiB2 and the inclusion bodies were purified. The differences between denatured and non-denatured WhiB2 were analyzed by circular dichroism and nuclear magnetic resonance. Ferrous ion (Fe 2+ ) was used to restore the iron-sulfur cluster of WhiB2. The interaction between WhiB2 and the upstream promoter sequence of the WhiBMtb gene was analyzed by nuclear magnetic resonance. The tertiary structure of WhiB2 and interacting proteins were analyzed and protein structure alignment was performed based on bioinformatics. Results:The structure of the renatured WhiB2 was basically the same as that of the non-denatuous WhiB2. In addition, Fe 2+ could restore the iron-sulfur cluster of WhiB2. It was found that WhiB2 could bind to the upstream promoter sequence of the WhiBMtb/WhiB2Ms gene. Conclusions:Mtb WhiB2 played a key role in the pathogenesis of tuberculosis, which would contribute to future exploration of novel targets against Mtb.