Production and Characterization of a Functional Putidaredoxin Reductase-Putidaredoxin Covalent Complex

In the cytochrome P450cam-dependent. monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on Pdx were activated. The yield of the EDC-mediated cross-link depended on the Pdx variant used and the redox state of both partners, decreasing in the following order: Pdr(ox)-Pdx(ox) > Pdr(ox)-Pdx(red) >= Pdr(red)-Pd-red. The Pdr-Pdx C73S/C85S conjugate was purified and characterized. Compared to the equimolar mixture of intact Pdr and Pdx, the fusion protein was more efficient in electron transfer to cytochrome c and, in the presence of Saturating levels of P450cam, more effectively supported camphor hydroxylation. On the basis of our results, we conclude that (i) the cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, (ii) Molecular recognition between Pdr and Pdx is redox-controlled and assisted by the Glu72(Pdx)-Lys409(Pdr) charge-charge Interact ions, and (ill) the high specificity of the Pdr-Pdx Couple may be due to finely tuned Interactions it the protein-protein Interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.