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

Abstract

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 α-subunit (SCSα) in a noncovalent manner and enhances its enzymatic activity, thereby providing a new target for P_i activation in mitochondria. Coupling ³²P labeling of intact mitochondria with SDS gel electrophoresis revealed that ³²P labeling of SCSα was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS), we showed that this enhanced ³²P labeling resulted from a simple binding of ³²P, not covalent protein phosphorylation. The ability of SCSα to retain its ³²P 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 ³²P binding to SCSα was increased in substrate-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.