Abstract
Data are presented which indicate that the diffusion-based collisions of ubiquinone with its redox partners in the mitochondrial inner membrane are a rate-limiting step for maximum (uncoupled) rates of succinate-linked electron transport. Data were obtained from experimental analysis of a comparison of the apparent activation energies of lateral diffusion rates, collision frequencies, and electron transport rates in native and protein-diluted (phospholipid-enriched) inner membranes. Diffusion coefficients for Complex III (ubiquinol:cytochrome c oxidoreductase) and ubiquinone redox components were determined as a function of temperature using fluorescence recovery after photobleaching, and collision frequencies of appropriate redox partners were subsequently calculated. The data reveal that 1) the apparent activation energies for both diffusion and electron transport were highest in the native inner membrane and decreased with decreasing protein density, 2) the apparent activation energy for the diffusion step of ubiquinone made up the most significant portion of the activation energy for the overall kinetic activity, i.e. electron transport steps plus the diffusion steps, 3) the apparent activation energies for both diffusion and electron transport decreased in a proportionate manner as the membrane protein density was decreased, and 4) Arrhenius plots of the ratio of experimental electron transport productive collisions (turnovers) to calculated theoretically predicted, diffusion-based collisions for ubiquinone with its redox partners had little or no temperature dependence, indicating that as temperature increases, increases in electron transport rate are accounted for by the increases in diffusion-based collisions. These data support the Random Collision Model of mitochondrial electron transport in which the rates of diffusion and appropriate concentrations of redox components limit the maximum rates of electron transport in the inner membrane.
Highlights
Data are presented which indicate thadt iftfhuesion- ofredox components [1,2,3,4,5,6,7,8,9,10,11,12,13]
Energies for bothdiffusion and electron transportde- To determine a rate-limiting stepin the complex sequence creased in a proportionate manner as the membrane of consecutive reactions of mitochondrial electron transport, protein density was decreased, an4d) Arrhenius plots we have utilized the concepts put forth in the theory of rate of the ratioof experimental electron transport producp-rocesses [20, 21] that are derived in part from the theory of tive collisions to calculated theoretically predicted,diffusion-basedcollisions forubiquinone with its redox partners had little or no temperature dependence, indicating thatas temperature increases, increases in electron transport rateare accounted for by the increases in diffusion-based collisions
Experof mitochondrial electron transport based on a large number imentally, this was accomplished by measuring the temperaof observations on the structure of the mitochondrial inner ture dependences of the appropriate Ds determined by FRAP
Summary
Values were derived from Wu et al [29] and based on diffusion coefficients determined by FRAP in dimyristoylphosphatidylcholine multibilayers at andabove the main phase transition (-23 "C). 0.1 was determined by the method of Williams [38] using the coefficients of Schneider et al [23]. Protein concentrations were determined by the method of Lowry [39], and lipid phosphorus was determined by the Bartlettmethod [40]. VI from horse heart) was purchased from Sigma. Rhodamine isothiocyanate was obtained from Research Organics. Purified Complex I11 was the gift of Dr Tsoo King (Institute of Structural and Functional Studies, Philadelphia, PA)
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