Arginines 65 and 310 in putidaredoxin reductase are critical for interaction with putidaredoxin.
In this study, we test the functional validity of the recently determined crystal structure of a covalently linked putidaredoxin reductase (Pdr)-putidaredoxin (Pdx) complex. The structure predicts several surface residues in Pdr as important for complex formation and/or electron transfer (ET). The R65A, R310A, R310E, K339A, N384A, K387A, and K409A mutants of Pdr have been prepared and characterized, and the mutational effects on the kinetics of Pdx reduction during single and steady-state turnover have been assessed. Replacement of Asp384 was found to have no effect on the Pdr-Pdx interaction. The K339A, K387A, and K409A substitutions moderately inhibited the binding affinity and reduction of Pdx, whereas the R65A and R310A mutations lowered the interprotein ET rate by 20-30-fold without perturbing the Pdx association step. The charge reversal on Arg310 had the most profound effect and decreased both the Pdr-to-Pdx ET and partner binding affinity by 100- and 8-fold, respectively. Our findings support the structural data and suggest that (i) the X-ray model is biologically relevant, (ii) arginines 65 and 310 are the key elements required for the formation of a productive ET complex with Pdx, (iii) the C-terminal lysine cluster assists in Pdx docking by fine-tuning Pdr-Pdx interactions to achieve the optimal geometry between the redox centers, and (iv) the basic surface residues in Pdr-like ferredoxin reductases not only define specificity for the redox partner but also may facilitate its dissociation.
Sevrioukova
Irina F
IF
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA. sevrioui@uci.edu
Poulos
Thomas L
TL
D001120:Arginine / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D001120:Arginine; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase