Interaction between cytochrome b5 and hemoglobin: involvement of beta 66 (E10) and beta 95 (FG2) lysyl residues of hemoglobin.
In erythrocytes the reduction of oxidized hemoglobin (methemoglobin) is dependent upon an electron transport reaction between cytochrome b5 and methemoglobin. These two proteins are believed to form a complex whose bonding is principally determined by complementary charge interactions between acidic groups of cytochrome b5 and basic groups of hemoglobin. In order to refine this model, three surface lysyl hemoglobin variants--namely Hb N Baltimore beta 95 (FG2) Lys leads to Glu, Hb I Toulouse beta 66 (E10) Lys leads to Glu, and Hb I Philadelphia alpha 16 (A14) Lys leads to Glu--have been studied with respect to their reducibility and ability to bind cytochrome b5. In the two former variants, the substituted amino acids are located near the heme crevice; in the third one the substitution lies far from it. Substitutions of lysine for glutamic acid in positions beta 66 and beta 95 perturb the formation of the cytochrome b5--hemoglobin complex and result in a dramatic impairment of the cytochrome b5-mediated reduction, whereas the same mutation in position alpha 16 has no effect. We conclude that the lysine residues in positions beta 66 and beta 95 are directly involved in the binding of cytochrome b5. The three-dimensional structure of hemoglobin suggests that the cytochrome b5-binding domain of hemoglobin is constituted by four lysine residues surrounding the heme crevice in both alpha and beta chains. Similarities with other interacting hemoproteins are discussed.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003580:Cytochromes; D006418:Heme; D006455:Hemoglobins, Abnormal; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008706:Methemoglobin; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship
D003580:Cytochromes; D006455:Hemoglobins, Abnormal; D006418:Heme; D008706:Methemoglobin; D008239:Lysine