Fluorescence of tryptophan residues in firefly luciferases and enzyme-substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptoph an residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) wa s studied. The conserved Trp-417(419) residue was shown to be not accessibl e to charged particles, and positively and negatively charged amino acid re sidues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quen ching of the tryptophan fluorescence. The distance between the luciferin mo lecule and Trp-417(419) was calculated: 11-15 and 12-17 Angstrom for P. pyr alis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an alloste ric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP ) is proposed.