Thermodynamic Basis of Electron Transfer in Dihydroorotate Dehydrogenase B from Lactococcus lactis: Analysis by Potentiometry, EPR Spectroscopy, and ENDOR Spectroscopy

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• 作者：Al-Walid A. Mohsen ; Stephen E. J. Rigby ; Kaj Frank Jensen ; Andrew W. Munro ; and Nigel S. Scrutton
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：June 1, 2004
• 年：2004
• 卷：43
• 期：21
• 页码：6498 - 6510
• 全文大小：282K
• 年卷期：v.43,no.21(June 1, 2004)
• ISSN：1520-4995

文摘

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzesthe conversion of dihydroorotate to orotate and the reduction of NAD⁺. The enzyme is a dimer ofheterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDORspectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S centerand to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 ± 3 mV)was determined from analysis of absorption data at 540 nm, where absorption contributions from the twoflavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E₁) and semiquinone/hydroquinone (E₂) couples for the FMN (E₁ = -301 ± 6 mV; E₂ = -252 ± 8 mV) and FAD (E₁ =-312 ± 6 mV; E₂ = -297 ± 5 mV) were determined from analysis of spectral changes at 630 nm.Corresponding values for the midpoint reduction potentials for FMN (E₁ = -298 ± 4 mV; E₂ = -259± 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) areconsistent with the values determined for the FMN couples in DHODB. During reductive titration ofDHODB, small amounts of the neutral blue semiquinone are observed at ~630 nm, consistent with themeasured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODBidentifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of freeflavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral featuresobserved during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reductionpotentials determined using UV-visible spectroscopy and further identify an unusual EPR signal withvery small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to theformation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2Scenter. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that aredistinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2Scenter and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamicframework for electron transfer in this complex iron-sulfur flavoprotein.