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) > or = Pdr(red)-Pdx(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 interactions, and (iii) the high specificity of the Pdr-Pdx couple may be due to finely tuned interactions at the protein-protein interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.
Churbanova
Inna Y
IY
Department of Molecular Biology, University of California, Irvine, California 92697-3900, USA.
Poulos
Thomas L
TL
Sevrioukova
Irina F
IF
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000378:metabolism; D002234:Carbodiimides / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000378:metabolism
C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; D001426:Bacterial Proteins; D002234:Carbodiimides; D005288:Ferredoxins; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase