Dismantling and Rebuilding the Trisulfide Cofactor Demonstrates Its Essential Role in Human Sulfide Quinone Oxidoreductase

Abstract

Sulfide quinone oxidoreductase (SQR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQR revealed a sulfur atom bridging the SQR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic and computational modeling approaches. Cyanolysis of SQR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. Cyanolysis leads to reversible loss of SQR activity, which is restored in the presence of sulfide. We captured a crystallographic intermediate in SQR that provides clues as to how the oxidized state of the cysteines is preserved. Computational modeling and MD simulations revealed an ~10 5 -fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.

Acidity and nucleophilic reactivity of glutathione persulfide

“…These estimations are based on considerations for LMW species; as usually with enzymes, specific protein effects could lead to further acceleration factors. In fact, recent computational estimations suggest a rate increase of ~10 5 -fold for the first step if the active site of SQOR is in the trisulfide instead of the disulfide state (73).…”

The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter

“…Recently, trisulfide intermediate formation has been observed during the catalytic turnover of SQR, a sufide‐catabolizing enzyme which can use cyanide as cosubstrate. Particularly, it has been shown that this enzyme harbors in its active site a cysteine trisulfide and, according to the model proposed by the authors, cyanide interacts with the catalytic trisulfide and removes one sulfur atom by a cyanolysis reaction [ 289 ].…”

Hydrogen Sulfide Oxidation by Sulfide Quinone Oxidoreductase

“…The bridging sulfur in the human SQOR cysteine trisulfide is sensitive to cyanolysis, which was initially used to detect its presence biochemically [73] . Cyanolysis was shown to occur through nucleophilic addition of the cyanide to the trisulfide, forming a transient CT complex [89] . Subsequent decay of the CT complex leads to a disulfanyl‐methanimido thioate intermediate, which was trapped in crystallo .…”

“…Subsequent decay of the CT complex leads to a disulfanyl‐methanimido thioate intermediate, which was trapped in crystallo . The bridging sulfur is extracted from this intermediate in the presence of excess cyanide, preserving the oxidation state of the active site cysteines as a cyclized 201 Cys‐S‐N‐CHS‐ 379 Cys species in an inactive form of the enzyme [89] . Incubation of cyanide‐treated SQOR with sulfide reforms the resting trisulfide, reactivating the enzyme.…”

“…The cysteine trisulfide is more reactive than a cysteine disulfide both in terms of electrophilicity and leaving group potential. Computational modeling and molecular dynamics simulations estimate that the trisulfide configuration contributes an ∼10 5 ‐fold rate enhancement over a disulfide for the initial nucleophilic addition of sulfide, [89] accounting for much of the ∼10 7 ‐fold higher rate constant for sulfide addition to SQOR versus to a cysteine disulfide in solution [92] …”