A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine-diphosphate binding to subunit B of the A(1)A(O) adenosine triphosphate (ATP) synthase from Methanosarcina mazei Gö1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X-ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 A resolution, showing the substituted Trp-residue inside the predicted adenine-binding pocket. The cocrystallized ATP molecule could be trapped in a so-called transition nucleotide-binding state. The high resolution structure shows the phosphate residues of the ATP near the P-loop region (S150-E158) and its adenine ring forms pi-pi interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F(1)F(O) ATP synthases, is discussed in light of a transition nucleotide-binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A(3)B(3)- and A(3)B(R416W)(3)-subcomplex, composed of subunit A and the B subunit mutant R416W, of the A(1)A(O) ATP synthase is shown.