Abstract
The Vigna radiata H+-translocating pyrophosphatases (VrH+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, archaea, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing PPi, the product of anabolic reactions. Although the crystal structure of H+-PPase has been solved recently, the H+ translocation mechanism of H+-PPase is still unclear. Therefore, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to investigate the dynamic of H+-PPase between the resting (apo form), initiated (bound with substrate analogue) and transient states (bound with Pi). When proteins replaced hydrogen in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we determined the structural dynamic and conformational changes via the deuterium uptake. In the highly conserved substrate binding and exit regions, HDX on H+-PPase showed a compact conformation against deuterium exchange upon binding with substrate analogue and product. In addition, the exit region of proton channel exhibited a rapid-changed deuteration in the short time in the presence of phosphate. These results revealed more details about the mechanism of proton translocating by H+-PPase during PPi hydrolyzing, which are useful for biological and agricultural applications.