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The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

Mitochondrial oxidative damage from free radicals may be a factor underlying aging. We investigated whether long-term administration of physiological levels of melatonin, a direct free radical scavenger and indirect antioxidant, influences mitochondrial respiratory activity in liver of senescence-accelerated mice (SAM). Liver was obtained in the middle of dark period of the daily light:dark cycle from SAMP8, a strain of mice prone to accelerated senescence, and from SAMR1, a senescence-resistant strain, at 6 and 12 months of age. Respiratory control index (RCI), adenosine-5-diphosphate (ADP)/O ratio, State 3 respiration and dinitophenol (DNP)-dependent uncoupled respiration exhibited significant age-associated decreases in SAMP8. SAMP8 also showed significant age-associated reductions in respiratory chain complex I and IV activities. No age-related effects were found in these parameters in SAMR1. Daily oral melatonin administration (2 microg/mL of drinking fluid) beginning at 7 months of age significantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and complex I and IV activities in both mouse strains when they were 12 months old. These results reveal age-related reductions in mitochondrial function in SAM mice which are modified by melatonin; the most likely explanation for the corrective actions of melatonin relate to its antioxidative actions in mitochondria and other portions of the cell. The implication of the findings is that melatonin may be beneficial during aging as it reduces the deteriorative oxidative changes in mitochondria and other portions of the cell associated with advanced age.

A series of nitrophenolic electron-transport inhibitors (2-substituted 4,6-dinitrophenols) of rat liver mitochondrial cytochrome-bc1 complex and of photosystem II (QB site) of spinach thylakoids was synthesized. The structure/inhibitory-activity relationship was examined to elucidate differences in the three-dimensional structure of the quinone redox site in the two systems. These inhibitors occupy the ubiquinone redox site of cytochrome-bc1 complex competitively with natural ubiquinol, probably at a Qo reaction center. The inhibitory activity tended to increase with the length of the 2-substituent, which may correspond to the isoprenoid side chain of ubiquinone and plastoquinone, increased in both experimental systems. However, the strict structural requirements of the 2-substituent for binding to the ubiquinone or plastoquinone redox site were not identical. The alkyl substituents with a branching structure at the alpha-position to the benzene ring were favorable for inhibition of the cytochrome-bc1 complex, but not of photosystem II. Molecular-orbital calculations indicated that the main chain of 2-substituents with an alpha-branching structure was almost perpendicular to the benzene-ring plane because of steric congestion between the alpha-methyl and phenolic OH groups. The main chain of 2-substituents without an alpha-branching structure was flexible. Molecular-orbital studies indicated that ubiquinol was most stable when the portion of the isoprenoid side chain adjacent to the quinol ring was perpendicular to the quinol-ring plane, because of steric congestion by the vicinal OH and methyl groups. The side chain of plastoquinol was flexible because of the lack of a vicinal methyl group. Thus, the difference in the inhibitory activities between the two systems seemed to reflect the difference in the configuration of the isoprenoid side chain of ubiquinone and plastoquinone. These results suggested that the quinone redox site of the cytochrome-bc1 complex may recognize the configuration of the side chain near the quinone ring in the strict sense, whereas that of photosystem II (QB site) may recognize it in a loose sense.