Abstract
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.
Highlights
Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations
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
A biological source of polysulfides in mammals was not known until recently, when we demonstrated that hemeproteins such as hemoglobin and myoglobin can support the catalytic oxidation of H2S to thiosulfate and polysulfides (Fig. 1D) [11, 12]
Summary
Structure of Human Hemoglobin with Bound Sulfide—The crystal structure of human FeIII-Hb incubated with H2S was determined at 1.79 Å resolution (Fig. 2, Table 1). To verify that the extra density observed at the distal side of the iron is a sulfur atom (Fig. 2A), sulfur anomalous dispersion signals were collected at a 1.77-Å wavelength, and diffraction was recorded to 2.8 Å resolution. The sulfur anomalous difference map was calculated to locate sulfur atoms in hemoglobin. The sulfur anomalous signal overlaps with the electron density near the iron atom, confirming the presence of a sulfur ligand on the distal side of heme. The distance between the proximal HisF8 (His-87 in the ␣-subunit and His-92 in the -subunit) NE2 and the iron atoms is 2.3 Å in both the ␣- and -subunits (Fig. 2, C and D). The bond length between the iron and the sulfur atoms with unre-
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