FLAVIN FLUORESCENCE DYNAMICS AND PHOTOINDUCED ELECTRON-TRANSFER IN ESCHERICHIA-COLI GLUTATHIONE-REDUCTASE

Time-resolved polarized flavin fluorescence was used to study the acti ve site dynamics of Escherichia coli glutathione reductase (GR). Speci al consideration was given to the role of Tyr(177), Which blocks the a ccess to the NADPH binding-site in the crystal structure of the enzyme . By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for similar to 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenc hing rate, electron transfer from Y177 to the light-excited isoalloxaz ine part of flavin adenine dinucleotide (FAD) is proposed as the mecha nism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolari zation process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from inte rnal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the cl osely related enzyme dihydrolipoyl dehydrogenase. The fluorescence int ensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluct uations are likely to contribute to the nonexponential decay and the p robability of quenching by a specific site, the concept of conformatio nal substates need not be invoked to explain the heterogeneous fluores cence dynamics.