THE DYNAMICS OF PROTON-TRANSFER AT THE C-SIDE OF THE MITOCHONDRIAL-MEMBRANE - PICOSECOND AND MICROSECOND MEASUREMENTS

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-tris ulfonate), was introduced into the inner aqueous space of inside-out s ubmitochondrial particles (SMP). Upon initiation of respiration, the d ye recorded acidification of this space. Incorporation of high concent rations of the dye (approximately 100 nmol/mg of protein) had no effec t on the respiratory functions of the vesicles, nor on their capacity to execute DELTAmuH+-coupled reverse electron transfer. The respirator y control ratio (RCR) remained as high as RCR > 4. Pulse irradiation o f the dye caused photodissociation of the proton from the 8-hydroxy po sition. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecon d fluorimetry measured the dissociation of the proton from the excited dye molecule (PHIOH.), while microsecond spectroscopy followed the re action between the proton and the ground-state anion (PHIO-). Numerica l integration of the differential rate equations describes the diffusi on of protons in the perturbed system. The nanosecond measurements yie ld the physical characteristics of the aqueous phase that dissolves th e dye. The apparent dielectric constant of that space is rather low (e psilon = 20). The diffusion coefficient of the proton is 2.3 x 10(-5) cm2/s, and the activity of water is a(H2O) = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is ta ken up by the hydration layer of the lipids and proteins of the C side of the membrane. The microsecond dynamics measurements indicate that the rates of proton binding to the membrane surface components reach a n equilibrium within 60 mus. On the basis of these figures, we conclud e that, under physiological conditions, the inner space of submitochon drial vesicles is in a homogeneous state of protonation.