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 inte raction with Mg2+/Mn2+ cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies. For a ternary complex formed by a stoichiometric m ixture of TF1, Mn2+, and ADP, the ESEEM and HYSCORE data reveal a P-31 hype rfine interaction with Mn2+ (\A(3(1P))\ approximate to 5.20 MHz), significa ntly larger than that measured for the complex formed by Mn2+ and ADP in so lution (\A(P-31)\ approximate to 4.50 MHz). The Q-band EPR spectrum of the Mn . TF1 . ADP complex indicates that the Mn2+ binds in a slightly distorte d environment with \D\ approximate to 180 x 10(-4) cm(-1) and \E\ approxima te to 50 x 10(-4) cm(-1). The increased hyperfine coupling with P-31 in the presence of TF1 reflects the specific interaction between the central Mn2 and the ADP beta -phosphate, illustrating the role of the enzyme active si te in positioning the phosphate chain of the substrate for efficient cataly sis. Results with the ternary Mn . TF1 . ATP and Mn . TF1 . AMP-PNP complex es are interpreted in a similar way with two hyperfine couplings being reso lved for each complex (\A(P-31(beta))\ approximate to 4.60 MHz and \A(P-31( gamma))\ approximate to 5.90 MHz with ATP, and \A(P-31(beta))\ approximate to 4.20 MHz and \A(P-31 gamma)\ approximate to 5.40 MHz with AMP-PNP). In t hese complexes, the increased hyperfine coupling with P-31 gamma compared w ith P-31(beta) reflects the smaller Mn . . .P distance with the gamma -phos phate compared with the beta -phosphate as found in the crystal structure o f the analogous enzyme from mitochondria [3.53 vs 3.70 Angstrom (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 6 21-628)] and the different binding modes of the two phosphate groups. The E SEEM and HYSCORE data of a complex formed with Mn2+, ATP, and the isolated beta subunit show that the P-31 hyperfine coupling is close to that measure d in the absence of the protein, indicating a poorly structured nucleotide site in the isolated beta subunit in the presence of ATP. The inhibition da ta 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 st ate analogue namely Mg . TF1 . ADP . AlFx with the equilibrium dissociation constant K-D = 350 muM and rate constant k = 0.02 min(-1). The ESEEM and H YSCORE data obtained for an inhibited TF1 sample, Mn . TF1 . ADP . AlFx, co nfirm the formation of the transition state analogue with distinct spectros copic footprints that can be assigned to Mn . . . F-19 and Mn . . . Al-27 h yperfine interactions. The P-31(beta) hyperfine coupling that is measured i n the inhibited complex with the transition state analogue (\A(P-31(beta))\ approximate to 5.10 MHz) is intermediate between those measured in the pre sence of ADP and ATP and suggests an increase in the bond between Mn and th e P-beta from ADP upon formation of the transition state.