The Dynamics of Proton Transfer at the C Side of the Mitochondrial Membrane: Picosecond and Microsecond Measurements

A. B. Kotlyar*, N. Borovok, S. Kiryati, E. Nachliel, M. Gutman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-trisulfonate), was introduced into the inner aqueous space of inside-out submitochondrial particles (SMP). Upon initiation of respiration, the dye recorded acidification of this space. Incorporation of high concentrations of the dye (∼ 100 nmol/mg of protein) had no effect on the respiratory functions of the vesicles, nor on their capacity to execute ΔμH+-coupled reverse electron transfer. The respiratory control ratio (RCR) remained as high as RCR > 4. Pulse irradiation of the dye caused photodissociation of the proton from the 8-hydroxy position. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecond fluorimetry measured the dissociation of the proton from the excited dye molecule (ΦOH*), while microsecond spectroscopy followed the reaction between the proton and the ground-state anion (ΦO). Numerical integration of the differential rate equations describes the diffusion of protons in the perturbed system. The nanosecond measurements yield the physical characteristics of the aqueous phase that dissolves the dye. The apparent dielectric constant of that space is rather low (e = 20). The diffusion coefficient of the proton is 2.3 × 10-5 cm2/s, and the activity of water is aH2o = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is taken 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 an equilibrium within 60 μs. On the basis of these figures, we conclude that, under physiological conditions, the inner space of submitochondrial vesicles is in a homogeneous state of protonation.

Original languageEnglish
Pages (from-to)873-879
Number of pages7
JournalBiochemistry
Volume33
Issue number4
DOIs
StatePublished - 1 Feb 1994

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