Enzymology
Structural and Mechanistic Insights into Hemoglobin-catalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products*Mechanism of Hemoglobin-catalyzed H2S Oxidation

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Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal.

crystal structure
heme
hemoglobin
hydrogen sulfide
oxidation-reduction (redox)

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*

This work was supported in part by National Institutes of Health Grants GM112455 (to R. B.) and DK111465 (to U.-S. C). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The atomic coordinates and structure factors (code 5UCU) have been deposited in the Protein Data Bank (http://wwpdb.org/).

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The abbreviations used are:

    H2S

    hydrogen sulfide

    Cys-SSH

    cysteine persulfide

    CAM

    carbamidomethyl

    FeIII-Hb

    methemoglobin or ferric hemoglobin

    MSR

    methionine synthase reductase

    TCEP

    tris(2-carboxyethyl)phosphine.