Activation energies for the ATP-driven reversal of oxidative phosphorylation in submitochondrial particles

Abstract

The participation of energy transfer Factor B at the second phosphorylation site between cytochromes b and c 1 in the respiratory chain was tested spectroscopically at the wavelength pair 562–575 mμ with ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) as substrate and ATP as energy source for the reversal of oxidative phosphorylation. At temperatures above 30°, Factor B produced a distinct stimulation of the activity of ammonia particles but did not alter the extent of cytochrome b reduction. Below 30° no effect of Factor B could be observed. A similar though not completely matching biphasic thermal effect was also seen in the reversal of oxidative phosphorylation between NADH dehydrogenase and cytochrome b (site 1) promoted by Factor A or B with the urea and ammonia-EDTA particles, respectively. A phosphorylating electron transport particle from heavy mitochondria (ETP H) showed the same biphasic thermal respose in the ATP-driven NAD + reduction as well as in its oligomycin-sensitive ATPase activity without the addition of any factors. In all the above cases, there was a break in the Arrhenius plot around 30° except for the reversal of oxidative phosphorylation at site 1 in ETP H. The activation energies and their changes on supplementation with the energy transfer factors differed for the particles. Factor B had no effect on the activation energy of ammonia particles in the reversal of site 1 or 2 but simply increased the rate of the reversal. The effect of oligomycin at low levels in the reversal of site 1 phosphorylation with ammonia particles was similar to the effect of Factor B. It is concluded, in agreement with previous reports, that oligomycin and Factor B act in the same region of the energy transfer reactions. Factor A strikingly decreased the activation energy for the reversal of phosphorylation at site 1 below 31° in urea particles from 43.8 to 24.3 kcal/mole, supporting a structural role for the factor in stimulating oxidative phosphorylation. Above 31° the change was from 27.5 to 20.5 kcal/mole. Omission of dithiothreitol or addition of low levels of p-chloromercuriphenylsulfonate (PCMS) produced a decrease in the rate of reversal of site 1 phosphorylation in ETP H. Concomitant with this decrease a break was induced in the Arrhenius plot at about 28°. The activation energy above the break was between 11.8 and 12.8 kcal/mole, similar to the value for Factor B-supplemented ammonia particles in the same temperature range. Below the transition temperature, the activation energy was 24.6–26.2 kcal/mole. It is concluded from these studies that reconstitution of oxidative phosphorylation occurs best at temperatures between 30 and 43° possibly due to easier reassembly of the membrane structure at the higher temperatures.