Phosphate (P i ) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that P i controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F 1 F 0 -ATPase in heart and skeletal muscle mitochondria. In these studies the effect of P i was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. P i enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. P i oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of P i was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [P i ]. These results demonstrate that P i signaling results in the balanced modulation of oxidative phosphorylation, by influencing both ⌬G H ؉ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems. Phosphate (P i )1 is the substrate for the phosphorylation of ADP to ATP in oxidative phosphorylation. Because ADP and P i are generated by ATPases in the cytosol, the potential roles of ADP and P i as cytosolic feedback signaling molecules regulating the rate of ATP production was one of the first models of the cytosolic regulation of mitochondrial ATP production (1, 2).However, over the years it has become apparent that the cellular regulation of oxidative phosphorylation is a very complex control network, with numerous potential rate-limiting steps affected by a variety of signaling molecules, including ADP, P i , Ca 2ϩ , creatine, and Mg 2ϩ (3-7). This network results in the ability of tissues to change significantly the rate of ATP generation without significantly modifying the metabolic intermediates coupled to many other processes in the cell. This has been termed an energy metabolism homeostasis (8). Toward a better understanding of this regulatory network, the effects of each putative signaling molecule on oxidative phosphorylation need to be characterized. The purpose of the current work was to further evaluate the effects of P i on different regulatory sites of oxidative phosphorylation in cardiac mitochondria.Phosphate is believed to enter cardiac mitochondria via a neutral phosphate transporter (P t ) in exchange for OH Ϫ or by co-transport with H ϩ (9). Thus, P i transport is linked to the mitochondrial inner membrane pH gradient (⌬pH m ) and the phosphate concentration gradient but not to the membrane potential (⌬⌿). Although P i transport has not been ascribed as a rate-limiting step for phosphate utilization in oxidative phosphorylation, this particular aspect of phosphate metabolism has not been extensively studied, espec...

The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.