Characterisation of the electron transfer and complex formation between flavodoxin from D. vulgaris and the haem domain of cytochrome P450 BM3 from B. megaterium.

Investigation of the complex formation and electron transfer kinetics between P450 BMP and flavodoxin was carried out following the suggested involvement of flavodoxin in modulating the electron transfer to BMP in artificial redox chains bound to an electrode surface. While electron transfer measurements show the formation of a tightly bound complex, the NMR data indicate the formation of shortly lived complexes. The measured k(obs) ranged from 24.2 s(-1) to 44.1 s(-1) with k(on) ranging from 0.07 x 10(6) to 1.1 x 10(6) s(-1) M(-1) and K(d) ranging from 300 microM to 24 microM in buffers of different ionic strength. This apparent contradiction is due to the existence of two events in the complex formation prior to electron transfer. A stable complex is initially formed. Within such tightly bound complex, flavodoxin rocks rapidly between different positions. The rocking of the bound flavodoxin between several different orientations gives rise to the transient complexes in fast exchange as observed in the NMR experiments. Docking simulations with two different approaches support the theory that there is no highly specific orientation in the complex, but instead one side of the flavodoxin binds the P450 with high overall affinity but with a number of different orientations. The level of functionality of each orientation is dependent on the distance between cofactors, which can vary between 8 and 25 A, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.

Improving catalytic properties of P450 BM3 haem domain electrodes by molecular Lego.

In this work the catalytic properties of a cytochrome P450 immobilised onto an electrode surface are improved by means of the molecular Lego approach.

Proton-coupled electron transfer of flavodoxin immobilized on nanostructured tin dioxide electrodes: thermodynamics versus kinetics control of protein redox function.

In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO(2) electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein's flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/semiquinone and semiquinone/hydroquinone midpoint potentials (E(q/sq) and E(sq/hq)) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. E(q/sq) exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s(-)(1) at pH 10 to 0.0074 s(-)(1) at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone.