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

Abstract

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.

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

“…This inference is consistent with the observation of two different types of complexes of rabbit microsomal cytochrome P450 2B4 with CPR, only one of which is governed by electrostatic interactions, whereas the second contact region is hydrophobic [59]. Our observation is also consistent with the recent indication of multiple orientations of the flavoprotein in the complexes of the heme domain of P450BM-3 with the flavodoxin from Desulfovibrio vulgaris (which served as a model of the FMN domain of BMR) [60]. …”

P450BM3(CYP102A1): connecting the dots

“…Electron transfer rates doubled in a flavodoxin/BMP construct as KCl concentrations were raised from 50 mM to 250 mM, for instance, underpinned by a significant tightening in binding between the two proteins, but then decreased on a further increase to 400 mM. 230,231 It was postulated that constructive reorganisation of the electrostatic complex accounted for the initial response, but that dissociation took place when ionic strength became excessive, as observed with other redox partner systems. 231 The N-demethylation of erythromycin by three separate fusions also displayed ionic strength-dependence across the 0-500 mM NaCl concentration range, peaking at 500 mM.…”

“…230,231 It was postulated that constructive reorganisation of the electrostatic complex accounted for the initial response, but that dissociation took place when ionic strength became excessive, as observed with other redox partner systems. 231 The N-demethylation of erythromycin by three separate fusions also displayed ionic strength-dependence across the 0-500 mM NaCl concentration range, peaking at 500 mM. 189 Kinetic titrations with palmitic acid became sigmoidal as ionic strength was reduced, the point at which Michaelis-Menten kinetics ceased to apply being higher in Tris/HCl (50 mM) than in phosphate (25 mM).…”

“…Detailed studies of the interactions between the two partners in the latter hybrid have been carried out. 231 Other microsomal P450 domains have been fused to BMR, allowing the in vitro oxidation of substrates oxidised by mammalian P450s. 189,392,393 Approximately 10% oxygenase activity was retained when BMR was fused to the NOS haem domain.…”

The electron transfer complex between nitrous oxide reductase and its electron donors

“…BiGGER has been used in the last decade to obtain structural models of several electron transfer and proteinprotein complexes [8][9][10], in most cases when the threedimensional structures of the proteins involved are available and the residues at the interface are either not known or known only for one of the partners. Although the coordinates used are considered as ''rigid bodies'', the algorithm offers an option called ''soft-docking'' that takes into account the conformational freedom of some of the surface residue side chains, such as lysine, to assist the prediction of the mode of binding of the two proteins.…”