Evidence for changes in the nucleotide conformation in the active site of H⁺-ATPase as determined by pulsed EPR spectroscopy

The conformation of di- and triphosphate nucleosides in the active site of ATPsynthase (H⁺-ATPase) from thermophilic Bacillus PS3 (TF1) and their interaction with Mg²⁺/Mn²⁺ cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies. For a ternary complex formed by a stoichiometric mixture of TF1, Mn²⁺, and ADP, the ESEEM and HYSCORE data reveal a ³¹P hyperfine interaction with Mn²⁺ (|A(³¹P)| ≃ 5.20 MHz), significantly larger than that measured for the complex formed by Mn²⁺ and ADP in solution (|A(³¹P)| ≃ 4.50 MHz). The Q-band EPR spectrum of the Mn· TF1·ADP complex indicates that the Mn²⁺ binds in a slightly distorted environment with |D| ≃ 180 x 10^-4 cm^-1 and |E| ≃ 50 x 10^-4 cm^-1. The increased hyperfine coupling with ³¹P in the presence of TF1 reflects the specific interaction between the central Mn²⁺ and the ADP β-phosphate, illustrating the role of the enzyme active site in positioning the phosphate chain of the substrate for efficient catalysis. Results with the ternary Mn·TF1·ATP and Mn·TF1·AMP - PNP complexes are interpreted in a similar way with two hyperfine couplings being resolved for each complex (|A(³¹P(β))| ≃ 4.60 MHz and |A(³¹P(γ))| ≃ 5.90 MHz with ATP, and |A(³¹P(β))| ≃ 4.20 MHz and |A(³¹P(γ))| ≃ 5.40 MHz with AMP - PNP). In these complexes, the increased hyperfine coupling with ³¹P(γ) compared with ³¹P(β) reflects the smaller Mn···P distance with the γ-phosphate compared with the β-phosphate as found in the crystal structure of the analogous enzyme from mitochondria [3.53 vs 3.70 Å (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628)] and the different binding modes of the two phosphate groups. The ESEEM and HYSCORE data of a complex formed with Mn²⁺, ATP, and the isolated β subunit show that the ³¹P hyperfine coupling is close to that measured in the absence of the protein, indicating a poorly structured nucleotide site in the isolated β subunit in the presence of ATP. The inhibition data obtained for TF1 incubated in the presence of Mg²⁺, ADP, Al(NO₃)₃, and NaF indicate the formation of the inhibited complex with the transition state analogue namely Mg·TF1·ADP·A1F(x) with the equilibrium dissociation constant KD = 350 μM and rate constant k = 0.02 min^-1. The ESEEM and HYSCORE data obtained for an inhibited TF1 sample, Mn·TF1·ADP·A1F(x), confirm the formation of the transition state analogue with distinct spectroscopic footprints that can be assigned to Mn···¹⁹F and Mn···²⁷Al hyperfine interactions. The ³¹P(β) hyperfine coupling that is measured in the inhibited complex with the transition state analogue (|A(³¹P(β))| ≃ 5.10 MHz) is intermediate between those measured in the presence of ADP and ATP and suggests an increase in the bond between Mn and the P(β) from ADP upon formation of the transition state.