Buildup of hydrogen sulfide (HS), which functions as a signaling molecule but is toxic at high concentrations, is averted by its efficient oxidation by the mitochondrial sulfide oxidation pathway. The first step in this pathway is catalyzed by a flavoprotein, sulfide quinone oxidoreductase (SQR), which converts HS to a persulfide and transfers electrons to coenzyme Q via a flavin cofactor. All previous studies on human SQR have used detergent-solubilized protein. Here, we embedded human SQR in nanodiscs (SQR) and studied highly homogenous preparations by steady-state and rapid-kinetics techniques. SQR exhibited higher catalytic rates in its membranous environment than in its solubilized state. Stopped-flow spectroscopic data revealed that transfer of the sulfane sulfur from an SQR-bound cysteine persulfide intermediate to a small-molecule acceptor is the rate-limiting step. The physiological acceptor of sulfane sulfur from SQR has been the subject of controversy; we report that the kinetic analysis ofSQR is consistent with glutathione rather than sulfite being the predominant acceptor at physiologically relevant concentrations of the respective metabolites. The identity of the acceptor has an important bearing on how the sulfide oxidation pathway is organized. Our data are more consistent with the reaction sequence for sulfide oxidation being: HS → glutathione persulfide → sulfite → sulfate, than with a more convoluted route that would result if sulfite were the primary acceptor of sulfane sulfur. In summary, nanodisc-incorporated human SQR exhibits enhanced catalytic performance, and pre-steady-state kinetics characterization of the complete SQR catalytic cycle indicates that GSH serves as the physiologically relevant sulfur acceptor.

Among the iron-sulfur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulfur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulfur cluster biogenesis. Here we report that among the iron-sulfur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) center in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.

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Hydrogen sulfide (H2S) is an environmental toxin and a heritage of ancient microbial metabolism that has stimulated new interest following its discovery as a neuromodulator. While many physiological responses have been attributed to low H2S levels, higher levels inhibit complex IV in the electron transport chain. To prevent respiratory poisoning, a dedicated set of enzymes that make up the mitochondrial sulfide oxidation pathway exists to clear H2S. The committed step in this pathway is catalyzed by sulfide quinone oxidoreductase (SQOR), which couples sulfide oxidation to coenzyme Q10 reduction in the electron transport chain. The SQOR reaction prevents H2S accumulation and generates highly reactive persulfide species as products; these can be further oxidized or can modify cysteine residues in proteins by persulfidation. Here, we review the kinetic and structural characteristics of human SQOR, and how its unconventional redox cofactor configuration and substrate promiscuity lead to sulfide clearance and potentially expand the signaling potential of H2S. This dual role of SQOR makes it a promising target for H2S‐based therapeutics.