Project summary Hydrogen sulfide (H2S) plays important signaling roles in mammals and modulates neuroprotection, cardioprotection and inflammatory responses in the gastrointestinal system. Low steady-state levels of H2S are maintained to prevent toxicity, but must increase to allow signaling. Low H2S levels are maintained by the activity of a mitochondrial sulfide oxidation pathway. The first and committing step in this pathway is catalyzed by the flavoprotein, sulfide quinone oxidoreductase (SQR). Aberrant H2S levels are associated with a number of neurodegenerative, cardiovascular, and inflammatory diseases making SQR an attractive therapeutic target. SQR converts H2S to an enzyme-bound cysteine persulfide and then transfers the sulfane sulfur from the persulfide to a small-molecule acceptor. Concomitant with this latter step, a cysteine disulfide reforms in the SQR active site while the electrons from sulfide oxidation are relayed to the FAD cofactor, to coenzyme Q10 and thereon, to complex III in the electron transfer chain. Key features of the SQR reaction mechanism are unclear and are hampered by the limited solubility of this membrane-bound enzyme. We will address the gaps in our understanding of this critical enzyme for controlling H2S levels by addressing the following aims. (i) We will characterize the kinetics of human SQR in membrane nanodiscs. We will assess the catalytic efficiency of various small-molecules to function as persulfide acceptors and use kinetic simulations to identify the preferred acceptor at physiologically relevant concentrations. These studies will shape our understanding of the major SQR product that is fed to the next step in the sulfide oxidation pathway. (ii) We will investigate the detailed pre- steady kinetic mechanism of SQR using stopped-flow spectroscopy to identify catalytic intermediates and to evaluate their kinetic competence. We will identify the cysteine residues involved in persulfide and in charge transfer intermediate formation with FAD, as well as the roles of select active site residues in accelerating sulfide oxidation by a factor of over ten million compared to the reaction of H2S with disulfides in solution. (iii) We will harness the remarkable catalytic efficiency of SQR to develop a sulfide biosensor. For this, we will subject human SQR to random mutagenesis and selectively screen for mutants with enhanced activity in E. coli. We will fuse the hyperactive SQR obtained from this screen with a circularly permuted yellow fluorescent protein to couple the sulfide reactivity of SQR into a measurable fluorescence signal. The successful completion of the proposed studies will deepen our understanding of the human SQR reaction that is key to controlling H2S levels, lead to the development of a sulfide biosensor as an invaluable first step for detecting and quantifying sulfide levels in cells, and provide a foundation for therapeutic targeting of SQR for modulating H2S levels.