P27 Interaction of H2S with hemeproteins modulates the concentration of free sulfide compounds in solution and mediates H2S signaling

Abstract

Metalloproteins, particularly hemeproteins have the potential to mediate H2S signaling in cells. In fact, using the hemoglobin I (HbI) from the clam L. pectinata as a model we have previously suggested that H2S binds tightly to the ferric iron of hemeproteins and that its release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction [1] , [2] . At high H2S concentrations, reduction of the heme iron is stimulated by a second H2S molecule with the concomitant formation of ferrous heme and free polysulfides. To define the molecular details implicated in the electron transfer process and the role of distal residues in this process, the structural aspects associated with the heme-H2S moiety were further evaluated using several HbI mutants and X-ray absorption techniques (XAS). The dynamics of H2Swithin the HbI heme pocket were also evaluated by time-resolved absorption spectroscopy in the picosecond–nanosecond time range. XAS data shows that upon H2S binding to the HbI samples their absorption edges were red shifted by ∼1 eV with respect to the unliganded counterparts, indicating an increase of electron density on the ferric iron. Overall, the results suggest the formation of an “FeII–H2S” like derivative that coexists with the FeIII–H2S complex, upon H2S binding to rHbI and the HbI mutants. For HbI-H2S and the GlnE7His-H2S mutant, an H-bonding with the bound ligand can further modulate the FeII–H2S moiety of the intermediate (i.e. FeII–SH−), which can in principle facilitate interaction with a second H2S molecule, stimulating formation of the final deoxy and H2S2 species. Photo-excitation of the HbI FeIII–H2S complex resulted in photo-dissociation of the H2S from the ferric HbI, followed by fast H2S geminate rebinding with time constant τgem = 12 ± 1 ps. The fast rebinding reflects the high affinity of ferric HbI for H2S and supports the notion release of H2S is facilitated by another H2S molecule, which promotes heme reduction and polysulfide fromation. Since only one geminate rebinding is observed, we infer that there is no docking site for H2S within the heme and that only one energy barrier exists between solvent and heme pocket for sulfide binding. Thus, the rate-limmiting step for H2S releaseform ferric HbI is the diffusion of an aditional sulfide from the solvent to the heme pocket, which occurs at longer times scales. The fact that sulfide release in controlled by the rate of H2S diffusion from the solution to the vicinity of the heme, strongly suggest that hemeproteins modulate the concentration of free sulfide in the external environment, mediating in turn H2S signaling in cells. Disclosure: Nothing to disclose.