This study demonstrates that one of the sites of Pi regulation in mitochondrial metabolism is SCS. This was shown by monitoring the binding of 32P to SCSα within the intact mitochondrial matrix (), solubilized mitochondrial homogenates () and in purified BSCS () as well as by measuring SCS activity in vitro (Figures and ). The binding of 32P to SCSα in intact mitochondria was dependent on the mitochondrial energetic state, reflecting the concentration of matrix Pi. The ability of SCSα to bind 32P in the absence of an energy source and under the harsh denaturing conditions of SDS, revealed that 32P-labeling in 2D gel studies is not limited to covalent protein modifications. Additionally, the binding of Pi to SCS was associated with an activation of SCS activity both in mitochondrial homogenates and purified BSCS. Taken together, these data reveal that the non-covalent binding of Pi to SCS is regulatory and linked to the energized state of the matrix. The pivotal role that SCS plays in the metabolism of the mitochondria is outlined in . SCS plays a crucial role in the citric acid cycle, as the only site of substrate level phosphorylation within mitochondria, and as a component of ketone oxidation and heme synthesis.
Figure 8 Role of Succinyl-CoA Synthetase in Mitochondrial Energetics and Biosynthesis. Succinyl-CoA synthetase (SCS) is in pink and the pathways associated with the citric acid cycle are shown in light blue; ketone oxidation and succinyl-CoA acetoacetate CoA transferase (more ...)
It has previously been shown that Pi can directly activate citric acid cycle dehydrogenases, including α-ketoglutarate dehydrogenase (26
), NAD-isocitrate dehydrogenase (27
) and malate dehydrogenase (28
) as well as several sites of oxidative phosphorylation (30
). The current study demonstrated that Pi activates the maximum velocity of SCS in a dose-dependent manner in mitochondrial homogenates and purified BSCS protein. This allosteric effect of Pi is a classical V-type allosteric activation associated with many multi-subunit enzymes, where the complexes are stablized and both the forward and reverse maximum velocities are increased by allosteric factors (41
). Since Pi has been shown to stablize the SCS complex (8
) and increase the Vmax of the reverse reaction, it is reasonable to assume that the forward reaction kinetics are also enhanced by Pi as found in other V-type interactions.
These results suggest that SCS is another target for Pi-activation in mitochondrial energetics. Further support for a Pi-induced activation of SCS is found in the study by Siess et al (29
), who determined the effects of Pi on the matrix metabolite concentrations of α-ketoglutarate, succinyl-CoA and malate in liver mitochondria. It was shown that Pi decreased matrix [α-ketoglutarate] and [succinyl-CoA] while increasing [malate]. This metabolite “cross-over” is consistent, but not unique, to an activation of SCS by Pi (29
). Also consistent with the activation of SCS in low-energy states is the ~2.5-fold increase in the SCS product, succinate, during cardiac ischemia (16
). Collectively, these results provide strong evidence that SCS activity is increased in the presence of mM Pi levels.
The Pi-induced activation of SCS is especially interesting in the functional context of myocardial ischemia or any energy supply-and-demand mismatch that occurs in the heart. The most rapid and large change in cytosolic metabolites that occurs during such an energetic-mismatch in the heart involves cytosolic [Pi] (44
). Thus, Pi is an excellent signaling molecule that is capable of activating the energy-conversion processes of the mitochondria as well as potentially supporting non-oxygen dependent substrate-level ATP formation. Under energy-limited conditions, such as myocardial ischemia or hypoxia, ATP production by anaerobic glycolysis can prevent broad cellular injury and maintain energization of the mitochondrial matrix by driving Complex V backwards as an ATPase (45
). However, during prolonged myocardial ischemia, anaerobic glycolysis is suppressed (47
), and therefore, anaerobic pathways of mitochondrial metabolism (i.e., substrate-level phosphorylation via SCS) are triggered to support ATP production and potentially mollify cardiac injury (16
). The increased activity of substrate-level phosphorylation via SCS has been suggested as the source of high succinate in the ischemic heart as discussed above. To support this notion, kidney proximal tubules subjected to hypoxia and re-oxygenation demonstrated that anaerobic metabolism of α-ketoglutarate plus aspartate increased the recovery of cellular ATP by intra-mitochondrial substrate-level phosphorylation at the level of SCS (17
). Therefore, we speculate that Pi acts as both a substrate and allosteric activator of substrate-level phosphorylation via SCS to generate matrix ATP that may limit ischemically-induced mitochondrial dysfunction. We did not attempt to directly monitor the allosteric effects of Pi on matrix ATP at anoxia because the interpretation of such studies is complicated by the fact that Pi is also a substrate for the forward (ATP synthetic) reaction. Thus, unraveling the different effects of Pi as a substrate and/or allosteric modulator of SCS in intact mitochondria will be difficult.
Direct chemical activity measurements of Pi within the mitochondrial matrix of intact cells or even isolated mitochondria are not currently available. Since the pH gradient is small in heart mitochondria (30
), it is likely that the driving force for the inner membrane transport of Pi is generated by a concentration gradient, where matrix [Pi] is much lower than cytosolic [Pi]. Cytosolic [Pi] in the heart increases with oxygen delivery limitations, as discussed above, or near maximum work levels as detected by 31
P NMR (49
). In skeletal muscle, cytosolic [Pi] increases more proportionally with workload, but also exists at a considerably higher concentration than heart in the resting state (50
). Thus, at the current time, the relationship between cytosolic [Pi] and workload with Pi-activity in the mitochondrial matrix is unknown. Furthermore, whether the Pi-induced activation process detected here is involved during normal work transitions in heart and skeletal muscle is also unknown.
Another unique finding of this study was that the non-covalent binding of 32
P to SCSα survived the entire SDS gel electrophoresis process. Notably, SCSα was the only mitochondrial protein in this study that bound 32
P in a manner that persisted throughout SDS gel electrophoresis. Given the harsh denaturing properties of SDS, it is generally assumed that non-covalent protein modifications do not persist. Thus, like previous reports (51
), we initially assumed that 32
P-labeling of SCSα resulted from phosphorylation. However, the ability to selectively dilute SCSα’s 32
P-incorporation with excess cold Pi, in the absence of a high energy nucleotide, implied that a majority of SCSα’s 32
P-association resulted from simple Pi-binding, and not phosphorylation. Furthermore, the labile nature of 32
P-binding to SCSα in the native protein (), coupled with the inability to exchange out its 32
P after SDS gel electrophoresis, suggested that conformational changes to SCSα by SDS trap Pi within the protein. Predictably, this extremely tight, high affinity binding of 32
P to SCSα allowed the label to persist throughout the SDS gel electrophoresis process.
The current study also revealed a pH-sensitivity for SCSα’s 32
P-binding, as acid-treatment decreased the intensity of labeling. This is likely due to changes in the charge state of phosphate or the SCS binding site as a function of pH. Since mitochondria have a bacterial ancestry, several studies have used acid-lability to screen for histidine phosphorylations in mitochondria. To date, SCSα is one of the few defined histidine phosphorylations in eukaryotes (52
). Although the decrease in 32
P-labeling may result from alterations to the phosphate ion or conformational changes to SCSα, this pH-sensitivity is important to consider when evaluating differences in the “phosphorylation” of SCSα, since such changes may also result from non-covalent Pi-binding.
The apparent affinity for Pi in the activation of SCS was found to be in the mM range (Figures and ). However, Pi was found to bind SCSα in the 32P gel studies despite the fact that only tracer amounts of Pi were added in order to keep the specific activity of 32P high. As described above, since 32P-binding to SCSα persists throughout SDS gel electrophoresis, the interaction is one of high affinity, which is inconsistent with the apparent mM affinity revealed in the activity assays. This apparent discrepancy is resolved by two observations. First, as shown in only a small fraction of SCSα's 32P-labeling is resolved upon exposure to SDS in the 2D BN-PAGE studies. Consequently, even if the overall affinity for SCS is the mM region, the high sensitivity of 32P autoradiography detects only a small fraction of the associated Pi in the native protein. Additionally, we found that the 32P could be exchanged out of the native protein but not the SDS treated protein in the gel. These later data suggest that denaturing of the protein traps phosphate in a high affinity site, thereby permitting its detection through the SDS PAGE process. These observations imply that while in-gel 32P-binding studies reflect the association of Pi with SCSα, this approach cannot be used to determine the mole fraction SCSα containing bound Pi.
The current study revealed a specific non-covalent binding of 32P to SCSα in substrate-depleted mitochondria. It is important to point out that the specific activity of 32P in the matrix is predictably different in energized and substrate-depleted mitochondria. When energized with carbon substrates, the matrix phosphorylation potential is high, resulting in a low matrix [Pi] and increasing the potential specific activity of 32P in the matrix Pi pool, thereby increasing the sensitivity of 32P-binding. However, in the substrate-depleted matrix, [Pi] is predictably higher, resulting in a lower matrix 32P specific activity, which decreases the 32P-binding sensitivity. Thus, the enhanced 32P-labeling of SCSα in substrate-depleted mitochondria occurred despite the predicted decrease in specific activity, underlining the fact that Pi association to SCSα is increased in the energy-limited state.
The enhanced 32
P-binding to SCSα observed in the substrate-depleted matrix suggests that phosphate binding to SCS could be a marker of compromised energy conversion in mitochondria with high matrix [Pi]. Several 31
P-NMR studies have described a large, NMR-invisible Pi-pool in mitochondria (~60% (53
)), believed to result from Pi that is bound to proteins or membranes (53
). Could the binding of Pi to SCS contribute to this NMR invisible pool of Pi? Our 2D gels estimate SCSα to be at a concentration of 0.27 nmol per mg mitochondria, by comparing the SCSα intensity with Complex IV. Assuming 2 μl of matrix volume per mg, the concentration of SCS is on the order of 0.2 mM, which could be quite significant relative to the low matrix [Pi] in the energized state (see above). The BN-PAGE analysis of the entire mitochondrial proteome also suggests that Complex V could be a significant binding site of matrix Pi under energized conditions (). However, in the energy-limited matrix, [Pi] is well above [SCS], and SCS is therefore not likely to significantly influence the chemical activity of Pi. Thus, the binding of Pi by SCS under energy-limited conditions may primarily serve to activate substrate-level phosphorylation.
In summary, Pi was shown to bind and allosterically enhance SCS activity. In the context of energy supply/utilization mismatched conditions, such as ischemia and hypoxia, the findings presented here suggest that a Pi-induced activation of SCS may anaerobically generate ATP in the matrix in order to minimize mitochondrial dysfunction and enable cellular repair before the onset of irreversible injury. The role of this activation process during normal work transitions in the heart and skeletal muscle is yet to be resolved.