BINDING OF THE OXIDIZED, REDUCED, AND RADICAL FLAVIN SPECIES TO CHORISMATE SYNTHASE - AN INVESTIGATION BY SPECTROPHOTOMETRY, FLUOROMETRY, AND ELECTRON-PARAMAGNETIC-RESONANCE AND ELECTRON-NUCLEAR DOUBLE-RESONANCE SPECTROSCOPY

Chorismate synthase (EC 4.6.1.4) binds oxidized ribonavin-5'-phosphate mononucleotide (FMN) with a K-D of 30 mu M at 25 degrees C, but in th e presence of 5-enolpyruvylshikimate-3-phosphate (EPSP), the K-D decre ases to ca. 20 nM. Similar effects occur with the substrate analogue ( 6R)-6-fluoro-EPSP (K-D = 36 nM) and chorismate (K-D = 540 nM). Fluores cence of oxidized FMN is slightly quenched in the presence of chorisma te synthase. Addition of EPSP or the (6R)6-fluoro analogue causes a sh ift of the fluorescence from 520 to 495 nm. Chorismate causes no shift in, but a quenching of, the fluorescence emission maximum. In the pre sence of EPSP, (6R)-6-fluoro-EPSP, or chorismate, the neutral flavinse miquinone is generated. The electron paramagnetic resonance (EPR) Line width of the flavin radical is indicative of a neutral flavinsemiquin one. Frozen solution electron nuclear double resonance (ENDOR) of the radical with (6R)-6-fluoro-EPSP shows a number of proton ENDOR line pa irs. The largest splitting is assigned to a hyperfine coupling to the methyl group beta-protons at position 8 of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A(perpendicular to ) = 8.07 MHz and A(parallel to) = 9.60 MHz, giving A(iso) of 8.58 MHz, consistent with a neutral semiquinone form. The isotropic hfc couplin g of the 8-methyl protons with (6R)6-fluoro-EPSP decreases by about 0. 5 MHz when chorismate is bound, indicating that the spin density distr ibution within the isoalloxazine ring system depends critically on the nature of the ligand. The redox potential of FMN in the presence of c horismate synthase was 95 mV more positive than that of free FMN (at p H 7.0), equivalent to a 1660-fold tighter binding of reduced FMN. The pH dependence of the redox potential of chorismate synthase-bound FMN exhibits a slope of -30 mV per pH unit between pH 6 and 9, indicating that the two-electron reduction of the flavin is associated with the u ptake of one proton; this, and the UV-visible spectrum, is consistent with the reduced flavin being bound to chorismate synthase in its mono anionic form.