Femtosecond Fluorescence Dynamics of Flavoproteins: Comparative Studies on Flavodoxin, Its Site-Directed Mutants, and Riboflavin Binding Protein Regarding Ultrafast Electron Transfer in Protein Nanospaces

Abstract

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.