Role of charge and fluidity in the incorporation of cytochrome oxidase into liposomes.

Inactive, lipid-depleted cytochrome oxidase was rapidly activated upon incubation with either neutral, basic, or acidic liposomes. When incubated with 0.5 to 10 times its weight in liposomes, the enzyme bound 20% of its weight in lipids. This amount of bound lipids probably corresponds to the “annular lipids” postulated to be tightly bound to cytochrome oxidase (Jest, P. C., Griffith, 0. H., Capaldi, R. A., and Vanderkooi, G. (1973). Proc. Natl. Acad. Sci. U. S. A. 70, 480-484). Cytochrome oxidase was functionally incorporated into liposomes, when incubated with more than 15 times its weight in either acidic or neutral liposomes containing both acidic and basic lipids. However, it bound only approximately its own weight in lipids. When functionally incorporated, the enzyme was oriented across the liposome membrane, and its activity was subject to respiratory control. Cholesterol and saturated phospholipids did not inhibit the binding of the enzyme to the liposomes, but did prevent its functional incorporation. Enzyme incubated with neutral liposomes containing only phosphatidylcholine replenished its annular lipids but did not bind to intact liposomes. Its activity was not under respiratory control. Cytochrome oxidase, whether incorporated into acidic or basic liposomes, was oriented unidirectionally with its cytochrome c binding moiety exposed. Subunits II and III were the most exposed to surface labeling with p-diazonium benzene[““Slsulfonate. Incorporation of the enzyme into basic liposomes partially denatured it, exposing also the hydrophobic Subunit I. Thus, the charge of the liposomes seems to play a role in the initial binding of the enzyme to the liposome but plays no role in the orientation of the enzyme in the liposome. We propose the following sequence for the incorporation of cytochrome oxidase into liposomes: (a) Formation of the protein. annular lipids complex, (6) binding of the above complex to the liposomes, (c) orientation of the complex with its more hydrophobic pole next to the liposomes, and Cd) penetration of the protein’annular lipids complex into the liposome membrane.