Effect of Nucleotides on the Thermal Stability and on the Deuteration Kinetics of the Thermophilic F 0 F 1 ATP Synthase

Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F 0 F 1 ,‐type ATP synthase from the thermophilic Bacillus PS3 (TF 0 F 1 ). The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF 0 F 1 , in the absence of nucleotides. The thermal denaturation occurs at a transition temperature (t m ) of 81.7°C. The remarkable thermostability of this enzyme was decreased upon tight binding of Mg 2+ . ATP to noncatalytic sites, whereas binding of Mg 2+ . ADP increased the temperature at which thermal denaturation occurred. At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding. With the aim to correlate these thermal effects with possible structural differences among the various forms of TF 0 F 1 , Fourier transform infrared spectroscopy was carried out. Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy. As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg 2+ . ATP than in the presence of Mg 2+ . ADP. Therefore, consistent with microcalorimetric data, binding of Mg 2+ . ADP induces conformational changes which shield amide protons to more buried hydrogen‐bonded structures, whereas binding of Mg 2+ . ATP results in a more open or flexible structure.