Succinyl-CoA Synthetase Is a Phosphate Target for the Activation of Mitochondrial Metabolism

Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism, and heme synthesis. Inorganic phosphate (P-i) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study, it was shown that P-i binds the porcine heart SCS alpha-subunit (SCS alpha) in a noncovalent manner and enhances its enzymatic activity, thereby providing a new target for P-i activation in mitochondria. Coupling P-32 labeling of intact mitochondria with SDS gel electrophoresis revealed that P-32 labeling of SCS alpha was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS), we showed that this enhanced P-32 labeling resulted from a simple binding P-32, not covalent protein phosphorylation. The ability of SCS alpha to retain its P-32 throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a P-i-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with millimolar concentrations of P-i. Since the level of P-32 binding to SCS alpha was increased in subs trate-depleted mitochondria, where the matrix P-i concentration is increased, we conclude that SCS activation by P-i binding represents another mitochondrial target for the P-i-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria.