ELECTRONIC-ENERGY TRANSFER AND FLUORESCENCE QUENCHING IN THE ACTIVE-SITES OF MERCURIC REDUCTASE

The FAD-containing enzyme mercuric reductase has been studied by means of steady-state and time-resolved fluorescence spectroscopy. The fluorescence relation of the excited state of the isoalloxazine ring of FAD can be described by a sum of two exponential functions. The two lifetimes are not due a different lifetime of each of the two FAD molecules of mercuric reductase. The FAD molecules are quenched dynamically by a quencher that is not sensitive to the solvent viscosity. In vitro activation induces a dynamic quenching of fluorescence, while upon binding of NADP+ the FAD molecules are both statically and dynamically quenched. Time-resolved fluorescence anisotropy experiments of mercuric reductase in water show that the isoalloxazine ring probably undergoes a rapid and restricted vibrational motion of small amplitude. Electronic energy transfer occurs between the two FAD molecules at a rate of about 3.4 X 10(7) s-1. The angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor is 137 +/- 2-degrees (or 43 +/- 2-degrees). From previous X-ray data of glutathione reductase we find that the corresponding angle is 160-degrees. This suggests that the isoalloxazine rings of mercuric reductase and glutathione reductase are mutually tilted in slightly different ways.