Control of Proteoliposomal Cytochrome Oxidase: Normal and Inverted Orientations
Cytochrome c oxidase-containing proteoliposomes were prepared by sonication in the presence of cytochrome c and pyranine, followed by column chromatography to remove external c and pyranine. Externally-facing oxidase (60%) was then assayed using external cytochrome c, and internally-facing oxidase (40%) using membrane- permeable TMPD. External activity generates an FCCP-sensitive pH gradient, alkaline inside, associated with a valinomycin-stimulated uptake of K+ ions. In steady state, such vesicles maintain both pH gradient and membrane potential and cycle potassium both electrophoretically and electroneutrally. Valinomycin does not release full respiration, although it abolishes membrane potential under conditions in which the pH gradient does not exceed 0.5 pH units. At low concentrations, valinomycin is inhibitory. We conclude that pH gradient is more important than membrane potential in controlling the oxidase turnover.
Internal activity generates an internal TMPD+ steady state, as well as internal acidity. The former is increased by valinomycin or by external cyt. c, both of which reduce or alter the sign of the membrane potential. In the presence of ascorbate, TMPD and cyt. c, enzyme molecules of both orientations are working simultaneously in the same vesicles .
TMPD+ retention requires the uptake of an anion or loss of a cation to maintain electroneutrality. In the absence of a proton pump, therefore, internal alkalinization would be predicted despite the reversed orientation. The internal acidification observed experimentally suggests the operation of a steady state proton pump, moving H+ ions inwards in exchange for an electrophoretic steady state loss of K+.
KeywordsRespiratory Control Ratio Steady State Equation Submitochondrial Particle Negative Inside Internal Acidification
oxidized TMPD (Wurster’s blue)
respiratory control ratio
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