Binding of the Oxidized, Reduced, and Radical Flavin Species to Chorismate Synthase. An Investigation by Spectrophotometry, Fluorimetry, and Electron Paramagnetic Resonance and Electron Nuclear Double
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- 作者：Peter Macheroux ; Jan Petersen ; Stephen Bornemann ; David J. Lowe ; and Roger N. F. Thorneley
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：February 6, 1996
- 年：1996
- 卷：35
- 期：5
- 页码：1643 - 1652
- 全文大小：612K
- 年卷期：v.35,no.5(February 6, 1996)
- ISSN：1520-4995
文摘
Chorismate synthase (EC 4.6.1.4) binds oxidizedriboflavin-5'-phosphate mononucleotide (FMN)with a KD of 30 
M at 25 
C, but in thepresence of 5-enolpyruvylshikimate-3-phosphate (EPSP), theKDdecreases to ca. 20 nM. Similar effects occur with thesubstrate analogue (6R)-6-fluoro-EPSP(KD = 36nM) and chorismate (KD = 540 nM).Fluorescence of oxidized FMN is slightly quenched in thepresenceof chorismate synthase. Addition of EPSP or the(6R)-6-fluoro analogue causes a shift of thefluorescencefrom 520 to 495 nm. Chorismate causes no shift in, but a quenchingof, the fluorescence emissionmaximum. In the presence of EPSP, (6R)-6-fluoro-EPSP,or chorismate, the neutral flavinsemiquinoneis generated. The electron paramagnetic resonance (EPR) line widthof the flavin radical is indicative ofa neutral flavinsemiquinone. Frozen solution electron nucleardouble resonance (ENDOR) of the radicalwith (6R)-6-fluoro-EPSP shows a number of proton ENDOR linepairs. The largest splitting is assignedto a hyperfine coupling to the methyl group 
-protons at position 8of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A
=8.07 MHz and A
= 9.60 MHz, givingAiso of 8.58 MHz,consistent with a neutral semiquinone form. The isotropic hfccoupling of the 8-methyl protons with(6R)-6-fluoro-EPSP decreases by about 0.5 MHz whenchorismate is bound, indicating that the spin densitydistribution within the isoalloxazine ring system depends critically onthe nature of the ligand. The redoxpotential of FMN in the presence of chorismate synthase was 95 mV morepositive than that of free FMN(at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN.The pH dependence of the redoxpotential of chorismate synthase-bound FMN exhibits a slope of -30 mVper pH unit between pH 6 and9, indicating that the two-electron reduction of the flavin isassociated with the uptake of one proton; this,and the UV-visible spectrum, is consistent with the reduced flavinbeing bound to chorismate synthasein its monoanionic form.
M at 25 C, but in thepresence of 5-enolpyruvylshikimate-3-phosphate (EPSP), theKDdecreases to ca. 20 nM. Similar effects occur with thesubstrate analogue (6R)-6-fluoro-EPSP(KD = 36nM) and chorismate (KD = 540 nM).Fluorescence of oxidized FMN is slightly quenched in thepresenceof chorismate synthase. Addition of EPSP or the(6R)-6-fluoro analogue causes a shift of thefluorescencefrom 520 to 495 nm. Chorismate causes no shift in, but a quenchingof, the fluorescence emissionmaximum. In the presence of EPSP, (6R)-6-fluoro-EPSP,or chorismate, the neutral flavinsemiquinoneis generated. The electron paramagnetic resonance (EPR) line widthof the flavin radical is indicative ofa neutral flavinsemiquinone. Frozen solution electron nucleardouble resonance (ENDOR) of the radicalwith (6R)-6-fluoro-EPSP shows a number of proton ENDOR linepairs. The largest splitting is assignedto a hyperfine coupling to the methyl group -protons at position 8of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A =8.07 MHz and A = 9.60 MHz, givingAiso of 8.58 MHz,consistent with a neutral semiquinone form. The isotropic hfccoupling of the 8-methyl protons with(6R)-6-fluoro-EPSP decreases by about 0.5 MHz whenchorismate is bound, indicating that the spin densitydistribution within the isoalloxazine ring system depends critically onthe nature of the ligand. The redoxpotential of FMN in the presence of chorismate synthase was 95 mV morepositive than that of free FMN(at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN.The pH dependence of the redoxpotential of chorismate synthase-bound FMN exhibits a slope of -30 mVper pH unit between pH 6 and9, indicating that the two-electron reduction of the flavin isassociated with the uptake of one proton; this,and the UV-visible spectrum, is consistent with the reduced flavinbeing bound to chorismate synthasein its monoanionic form.