The Fluorescence Spectrum of the Introduced Tryptophans in the 3( F155W)3 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 F155W mutant of Escherichia coli F1 (bovine heart mitochondria F1 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. Chem. 271, 18711–18718). In contrast, addition of MgADP, Mg-5 -adenylyl , -imidophosphate (MgAMPPNP), and MgATP in 1:1 ratios to the 3( F155W)3 subcomplex of thermophilic Bacillus PS3 F1 (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 MgAMPPNP 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 F155W 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 F155W 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 subunits in the 3 3 subcomplex of TF1 can simultaneously exist in the completely closed conformation.