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

Abstract

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 , z los s-') the intermediate state decays to the stable semiquinone form of flavodoxin. The d C of the conformational change at pH 8 is about 29 kJ mol-(0.3 eV). This means that the upper limit for the pKof N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mo1-l (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 i 1) x lo8 M-s-5) mV.(PH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 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 < Em (Fld/FldH -) < -290 mV. The exact value will depend on the AG 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.Flavodoxin (from Megasphaera elsdeizii) is a negatively charged protein with a molecular mass of about 16000 Da, which contains FMN as the prosthetic group. Flavodoxin is assumed to be an electron donor and acceptor for hydrogenase and is formed in great quantities if the bacteria are grown in an iron-deficient medium. When iron is present, ferredoxin is formed. So in the obligatory hydrogen production of the bactcrium flavodoxin takes over the role of ferredoxin. M . elsdenii ferredoxin contains two [4Fe-4S] clusters.The oxidation-reduction properties of flavodoxin can be described by two one-electron steps which have different midpoint potentials. At pH 8 the redox behaviour is given by Eqns (1) and (2) [l].where Fld is the oxidized form of flavodoxin, FldH the halfreduced or semiquinone form and FldH-the fully reduced or hydroquinone form. For more detailed information see the excellent review article of Mayhew and Ludwig [l]. As was proposed by Muller et al. [2], the semiquinone form is probably stabilized by the protonation of the N-5 of the FMN and/or the subsequent formation of a hydrogen bond between this proton and the peptide backbone. In the semiquinone form of flavodoxin from Clostridium M P there is probably a hydrogenAhhreviutions. MV'', methyl viologen; BV", benzyl viologen; Fld, flavodoxin; F...

Influence of 8.alpha.-imidazole substitution of the FMN cofactor on the rate of electron transfer from the neutral semiquinones of two flavodoxins to cytochrome c

“…Finally, the results support and extend the previous pulse radiolysis work (van Leeuwen et al, 1983) and demonstrate that proton release during the redox reaction of flavodoxin semiquinone and a one-electron acceptor proceeds at a much faster rate than the processes associated with the electrontransfer event (the latter would include any bond distance or bond angle changes or any changes in solvation that occur during the formation of the transition state). The pH independence of the reaction rate constant implies that the difference in flavodoxin semiquinone redox potential between low and high pH does not affect the reaction kinetics, even though it has previously been shown (Meyer et al, 1983;Cheddar et al, 1986;Tollin et al, 1986) that, at a given pH, redox potential differences do exert a measurable influence on the apparent second-order rate constants for electron transfer.…”

“…These data constitute strong evidence that the electron-transfer step is not coupled to proton release from the neutral flavin semiquinone during le-oxidation; Le., proton release proceeds much more rapidly than electron transfer (see below for further comment). van Leeuwen et al (1983) have observed that the rate of Megasphaera elsdenii flavodoxin reduction to the semiquinone by the radical form of methylviologen is pH independent between pH 7.0 and 9.2. This observation is also consistent with a kinetic "uncoupling" of electron transfer and protonation of the flavin; Le., the rate of protonation is much faster than the rate of one-electron reduction.…”

Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN

“…The values determined for the midpoint redox potentials of the two steps in the reduction of the recombinant protein are very similar to the values reported from two laboratories for the native protein [18,54]. However, two other reports have given values for the semiquinone/hydroquinone couple ( E 1 ) that are about 20 mV more negative [29,55], and a measurement by pulse radiolysis gave a value of −410 ± 30 mV [56]. Vervoort et al .…”

Heterologous Ferredoxin Reductase and Flavodoxin Protect Cos-7 Cells from Oxidative Stress

“…Anyway, if non-processed MLS-Fld displays lower or no function, it is possible that the achieved levels of Fld activity are insufficient to cope with MV challenge. Regarding this point van Leeuwen et al [56] have reported that the reaction of MV radicals with Fld increases with Fld concentration and decreases when the concentration of oxidized viologen raises. In our studies we could be in the presence of low Fld (unprocessed protein) and high oxidized MV levels (high MV concentration imposed) explaining our observations.…”