Exploring the conformational equilibrium of E-coli thioredoxin reductase: Characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reduc tase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-reso lved fluorescence of the flavin cofactor in combination with circular dichr oism (both in the flavin fingerprint and far-UV regions) and steady-state f luorescence and absorption spectroscopy. The spectroscopic data show two co nformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measur ements make it possible to distinguish between the two different population s: Dominant picosecond lifetimes of similar to1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluor escence with two time constants in the range of 0.2-1 ns (total contributio n 17%) originates from enzyme molecules in the FR conformation. The near ab sence of fast lifetime components in oxidized wild-type TrxR supports the i dea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of th e FR conformation. Because of the large difference in fluorescence characte ristics, fluorescence measurements on time scales longer than 100 ps are fu lly determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme an d TrxR C138S stabilizes the enzymes in the FR conformation. Specific bindin g of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Ra ising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.