Orientation of the carboxyl and NH2 termini of the membrane-binding segment of cytochrome b5 on the same side of phospholipid bilayers.

The present data show that the carboxyl terminal end of the membrane binding segment (nonpolar peptide) of cytochrome b5 is present on the same side of phospholipid bilayers as the hydrophilic, heme-containing, NH2-terminal segment. This orientation was determined by observing rapid ionization of both tyrosyl residues at positions 5 and 8 from the carboxyl terminus upon addition of sodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. The rate of ionization of both aromatic residues occurred at least 1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration of our earlier characterization of cytochrome b5 structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer.