Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bond

Cystathionine beta-synthase (CBS) is the central enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of the three major enzymes involved in the biogenesis of H2S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a (CXXC275)-C-272 motif whose function has yet to be determined. In the present study, we demonstrated that the CXXC motif exists in oxidized and reduced states in the recombinant enzyme by mass spectroscopic analysis and a thiol labeling assay. The activity of reduced CBS is similar to 2-3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys(272-)Cys2(75) disulfide bond in CBS has a midpoint potential of -314 mV at pH 7.4. Additionally, the CXXC motif also exists in oxidized and reduced states in HEK293 cells under oxidative and reductive conditions, and stressing these cells with DTT results in more reduced enzyme and a concomitant increase in H2S production in live HEK293 cells as determined using a H2S fluorescent probe. By contrast, incubation of cells with aminooxy-acetic acid, an inhibitor of CBS and cystathionine gamma-lyase, eliminated the increase of H2S production after the cells were exposed to DTT. These findings indicate that CBS is post-translationally regulated by a redox-active disulfide bond in the CXXC motif. The results also demonstrate that CBS-derived H2S production is increased in cells under reductive stress conditions.