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.
Fantuzzi
Andrea
A
Division of Biomolecular Sciences, Imperial College London, SW7 2AZ London, UK.
Meharenna
Yergalem T
YT
Briscoe
Paul B
PB
Guerlesquin
Francoise
F
Sadeghi
Sheila J
SJ
Gilardi
Gianfranco
G
D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D004583:Electrons; D005418:Flavodoxin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D009994:Osmolar Concentration; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D005418:Flavodoxin; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases