The role of COOH-terminal anionic residues in binding cytochrome b5 to phospholipid vesicles and biological membranes.

The COOH-terminal, anionic residues of the membrane binding segment of cytochrome b5 were examined to determine their possible significance in stabilizing the "tight" binding of the cytochrome to phospholipid vesicles. The removal of the 6 COOH-terminal residues, which include the carboxyl groups of Glu 132 and Asn 133, by carboxypeptidase digestion resulted in the loss of the characteristic "tight" binding to either synthetic phospholipid vesicles or isolated microsomes. Chemical modification of the four carboxyl groups of the nonpolar peptide of cytochrome b5 with carbodiimide and methylamine to produce a derivative with no anionic charged residues also resulted in a loss of this type of stable membrane interaction. These results suggest that the short polar COOH-terminal segment, containing two of the four carboxyl groups of the membrane binding domain of cytochrome b5, plays a crucial role in lipid-protein interactions that lead to the normal "tight" binding both in situ and in reconstituted phospholipid bilayer systems.