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Potentiometric studies of native and flavin-substituted Old Yellow Enzyme
Abstract:
We have measured the redox potentials for the flavin cofactor of native Old Yellow Enzyme and for a series of chemically modified flavin derivatives bound to the apoprotein. These flavin derivatives have midpoint potentials ranging from -120 to -300 mV in free solution. For the native enzyme, the midpoint potential of the first one-electron couple EFMNox + e- in equilibrium EFMN-. is E1 = -245 +/- 5 mV and that for the second one-electron couple EFMN-. + e- in equilibrium EFMN red is E2 = -215 +/- 5 mV in 0.1 M phosphate buffer at pH 7.0 and 25 degrees C. Thus, the apoprotein lowers the two-electron midpoint potential of FMN below its value in free solution (Emid,bound = -230 mV, Emid,free = -210 mV). A similar effect on the two-electron midpoint potentials of most of the chemically modified flavins is observed upon binding to the apoprotein. Therefore, the relative order of the respective midpoint potentials of this series of flavins is maintained upon binding to the apoenzyme. However, the effect of the apoprotein on the separation between E1 and E2 varies considerably over this series of flavin derivatives, resulting in quite different levels of thermodynamic stability for the one-electron-reduced (semiquinone) forms of the flavin-substituted enzymes. The optical absorption spectra of these flavin-substituted enzymes were determined in the presence of several phenolic compounds which are known to bind to the native enzyme and to give characteristic long wavelength transitions. We have found that the positions of the wavelength maxima of these transitions are shifted to longer wavelengths as the measured redox potentials of the enzyme-bound flavins are increased. The relationship between the energy of these long wavelength transitions (expressed as vCT = 1/lambda max) and the redox potential of the enzyme-bound flavin is interpreted as further evidence that these transitions arise from charge-transfer complexes between the phenolic compounds and the flavin.