Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H2S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H2S toxicity. Single point mutations at E7, B10, and E11 were introduced into the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild-type recombinant proteins were first evaluated using the well-known ferrous CO and O2 derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O 2 binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, -PheB10Val, -PheB10Leu, and -E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H2S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H2S complexes, which show reduction of heme iron as judged by the appearance of the ν4 oxidation state marker at 1356 cm -1, indicative of heme-FeII species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting the strongest H-bonding interactions. Overall, the results presented here show that (a) H2S association is regulated by external kinetic barriers, (b) H2S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction, (c) at high H2S concentrations, reduction of the ferric center dominates, and (d) reduction of the heme is also enhanced in those HbI mutants having polar distal environments.
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