Characterization of a new three-component cytochrome P450 system from Thermobifida fusca

Abstract

Within this thesis, a new three component cytochrome P450 monooxygenase system from the moderately thermophilic bacterium Thermobifida fusca has been characterized biochemically, structurally and electrochemically. This system comprises the ferredoxin reductase FdR9, the ferredoxin Fdx8 as well as the P450 monooxygenase CYP222A1. Electrochemical characterization of anaerobically purified Fdx8 by Mossbauer spectroscopy, EPR and ICP-MS revealed the presence of a [3Fe–4S]1+-cluster with a histidine residue in place of a cysteine, that is found in [4Fe-4S]-cluster ferredoxins. Mutagenesis of this histidine to cysteine did not yield a [4Fe-4S]-cluster ferredoxin but prevented cluster incorporation. Moreover, cyclic voltammetry of Fdx8 indicated a positive redox potential for this ferredoxin, which is unusual for ferredoxins of P450 systems, but could be due to the presence of the histidine in the coordination region of the cluster. Protein-protein interaction studies of the members of the three-component system using MST, ITC and NMR confirmed binding of Fdx8 to FdR9 and CYP222A1 with KD values of 1 and 3 µM, respectively. ITC measurements further revealed that Fdx8-CYP222A1 as well as Fdx8-FdR9 binding is driven by hydrogen bonding and hydrophobic interactions. Moreover, thermodynamic parameters ?H and ?S indicated a temperature-dependent interaction for Fdx8-FdR9, which might imply conformational changes during binding. Possible conformational changes were also observed for Fdx8 during binding to CYP222A1 as indicated by protein NMR measurements. Structure determination by X-ray crystallography for all three components of the P450 system was also attempted. Crystals of FdR9 and CYP222A1 could be obtained and structures of both proteins have been determined at 1.90 and 1.85 A resolution, respectively, using molecular replacement. For Fdx8, however, no crystals could be obtained despite testing many different conditions. SEC-MALS measurements confirmed that Fdx8 is a monomer in solution. Moreover, Fdx8 structure determination was also attempted by protein NMR but was hampered due to the paramagnetic effect of the iron-sulfur cluster of the ferredoxin. Instead, a homology model has been generated. Finally, electron transfer within the three-component P450 system was studied using CO-difference spectroscopy and bioconversions. Unfortunately, investigation of the electron transfer from Fdx8 to CYP222A1 was hampered as no substrate of CYP222A1 could be identified so far.