Sulfide Oxidation and Signaling

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Hydrogen sulfide (H2S), a signaling molecule that elicits profound physiological effects, is a product of mammalian sulfur metabolism and is synthesized at relatively high rates. H2S is biosynthesized by three enzymes in the sulfur network of which two, cystathionine beta- synthase (CBS) and gamma-cystathionase, reside in the cytoplasmic transsulfuration pathway while the third, mercaptopyruvate sulfurtransferase, is involved in cysteine catabolism. Since H2S is highly toxic, cells avoid its build-up by an efficient oxidation pathway that is housed in mitochondria and coupled to the energy-generating electron transfer chain. The constituent proteins include sulfide-quinone oxidoreductase, a persulfide dioxygenase, rhodanese and sulfite oxidase. While our studies on H2S biogenesis are supported by HL58784, our work on H2S oxidation and signaling are supported by GM130183. It is becoming increasing clear that the biosynthetic and catabolic pathways for H2S interact and modulate each other. Therefore, the sulfide research supported by HL58784, scheduled to expire this year, will be folded into and then formally included in the competitive renewal of GM130183. In this supplemental project, the following specific aims will be addressed to elucidate fundamental mechanisms of regulation of H2S synthesis by CBS in normal and disease states: i) elucidate the steady state kinetics of linker mutants in CBS in the canonical transsulfuration and non-canonical H2S-generating reactions, (ii) investigate perturbations in the heme environment by EPR spectroscopy and potentiometric titrations, (iii) assess the impact of the mutations on AdoMet-dependent modulation of CO and NO• binding to ferrous heme and on the cellular flux of sulfur, and (iv) crystallize the linker mutants that appear to be less prone to aggregation compared to wild-type CBS. The impact of the proposed studies will be fundamental (i.e. elucidating mechanims of allosteric regulation at the level of CBS and in the pathway), medical (i.e. understanding the biochemical basis of failure of disease- causing CBS mutations), and most importantly, training a URM scientist of high promise.