P58 Hemoglobin I mutants from Lucina pectinata to study the role of the distal residues in the sulfheme formation

Abstract

The discovery that hydrogen sulfide is produce in mammalian tissues has increased dramatically the need to further understand the interactions of hydrogen sulfide with hemeproteins and hemeperoxidases. The human hemoglobin (LeuB10, HisE7, ValE11), myoglobin (LeuB10, HisE7, ValE11) and lactoperoxidase (Arg, HisE7), in the presence hydrogen peroxide (H2O2), and hydrogen sulfide (H2S), form an inactive sulfheme derivate characterized by a covalent sulfur modification of the heme “B” pyrrole ring. The derivate formation causes a partial inactivation of myoglobin, human hemoglobin and lactoperoxidase. However, the invertebrate organism Lucina pectinata remains unaffected in the presence of H2S and O2. The hemoglobin I (PheB10, GlnE7, PheE11) of this clam is the sulfide reactive protein that binds H2S as ligand. It has a glutamine in the distal E7 position instead of typical histidine found in hemoglobin, myoglobin and lactoperoxidase. Studies with hemeproteins demonstrate that the distal residues play an important role in the sulfheme derivates formation since the reactivity of H2S with hemeproteins are controlled by the polarity of the distal heme cavity, and the stereo orientation of the distal side residues. To confirm it, HbI mutants that mimic the active site of myoglobin and lactoperoxidase were done by site-directed mutagenesis. Then UV–vis spectroscopic analyses were made on Mb, Hb, HbI, and HbI mutants protein samples upon the reaction with hydrogen peroxide/oxygen and hydrogen sulfide. So far mutants with HisE7 apparently are the only mutants to show the characteristic UV–vis band at 624 nm that indicates the sulfheme product formation.