A pulse‐radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism

Pulse‐radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is –(480+30)mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change ( k 2 ∼ 10 5 s −1 ) the intermediate state decays to the stable semiquinone form of flavodoxin. The Δ G of the conformational change at pH 8 is about 29 kJ mol −1 (0.3 eV). This means that the upper limit for the p K of N‐5 in the semiquinone form will be 13. The activation energy of the conformational changes is 43 kJ mol −1 (0.45 eV). The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion‐controlled with a forward rate constant of (7 ± 1) × 10 8 M −1 s −1 (pH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be –(408 ± 5) mV. A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two‐electron process, the midpoint potential of which should be located between –440 mV < E m (Fld/FldH − ) < ‐290 mV. The exact value will depend on the ΔG of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two‐electron donor.