The fluorescence spectrum of the introduced tryptophans in the α3(β3F155W)3γ subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F, (bovine heart mitochondria F, residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. ChenL 271, 18711-18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,y-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F-1 (TF1) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF1 does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two 13 subunits in the alpha(3)beta(3)gamma subcomplex of TF1 can simultaneously exist in the completely closed conformation.