Characterisation of the electron transfer and complex formation between Flavodoxin from D. vulgaris

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• 作者：Andrea Fantuzzi ; Yergalem T. Meharenna ; Paul B. Briscoe ; Francoise Guerlesquin ; Sheila J. Sadeghi ; Gianfranco Gilardi
• 关键词：Electron transfer ; Flavodoxin ; P450 BMP ; Transient complex ; Protein– ; protein docking
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2009
• 出版时间：April 2009
• 年：2009
• 卷：1787
• 期：4
• 页码：234-241
• 全文大小：1296 K

文摘

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 × 10⁶ to 1.1 × 10⁶ s^− 1M^− 1 and K_d ranging from 300 μM to 24 μM 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 Å, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.