Mitochondrial ATP Synthase Catalytic Mechanism: A Novel Visual Comparative Structural Approach Emphasizes Pivotal Roles for Mg<sup>2+</sup> and P-Loop Residues in Making ATP

The mitochondrial ATP synthase (F<sub>o</sub>F<sub>1</sub>) is one of the most abundant, important, and complex enzymes found in animals and humans. In earlier studies, we used the photosensitive phosphate analogue vanadate (V<sub>i</sub>) to study the enzyme’s mechanism in the transition state. Significantly, these studies showed that Mg<sup>2+</sup> plays an important role in transition state formation during ATP synthesis. Additionally, in both MgADP·V<sub>i</sub>-F<sub>1</sub> and MgV<sub>i</sub>-F<sub>1</sub> complexes, photoactivation of orthovanadate (V<sub>i</sub>) induced cleavage at the third residue within the P-loop (GG<u>A</u>GVGKT), i.e., βA158, suggesting its proximity to the γ-phosphate during transition state formation. However, despite our recent release of the F<sub>1</sub>-ATPase structure containing V<sub>i</sub>, the structural details regarding the role of Mg<sup>2+</sup> have remained elusive. Therefore, in this study, we sought to improve our understanding of the essential role of Mg<sup>2+</sup> during transition state formation. We utilized Protein Data Bank structural data representing different conformational intermediates of key steps in ATP synthesis to assemble a database of positional relationships between landmark residues of the catalytic site and the bound ligand. Applying novel bioinformatics methods, we combined the resulting interatomic spatial data with an animated model of the catalytic site to visualize the exact nature of the changes in these positional relationships during ATP synthesis. The results of these studies reported here show that the absence of Mg<sup>2+</sup> results in migration of inorganic phosphate (P<sub>i</sub>) from βA158 to a more medial position in the P-loop binding pocket, thereby disrupting essential placement and orientation of the P<sub>i</sub> needed to form the transition state structure and therefore MgATP.