Normalized F344W-MDE fluorescence emission spectra in the presence of (A) ADP (+) and mant ADP (◊) or (B) ATP (+) and mant ATP (◊) relative to the apo state (○). Spectra are corrected for the fraction of F344W-MDE not bound to nucleotide. Quenching of 344W fluorescence is observed in the presence of each nucleotide: ADP, 81% apo; mant ADP, 76% apo; ATP, 76% apo; mant ATP, 58% apo. Quenching of fluorescence relative to the apo state in the presence of nucleotides lacking the mant moiety is due to changes in the 344W environment. Additional loss of intensity in the presence of mant nucleotides (e.g., mant ATP compared to ATP or mant ADP compared to ADP) is due directly to FRET between 344W and the mant moiety at the active site.