The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (16), p.16135-16142
• Main Authors: Kuznetsov, Vadim Yu, Blair, Emek, Farmer, Patrick J, Poulos, Thomas L, Pifferitti, Amanda, Sevrioukova, Irina F
• Format: Article
• Language: English
• Subjects: Ferredoxins - genetics; Ferredoxins - metabolism; Kinetics; Models, Molecular; Mutation; NADH, NADPH Oxidoreductases - metabolism; Oxidation-Reduction; Protein Structure, Tertiary; Pseudomonas putida; Pseudomonas putida - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M500771200

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 A. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp(38) and Trp(106), and at the edge of the protein-protein interface, Tyr(33) and Arg(66), were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Delta106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Delta106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp(106) and Tyr(33)/Arg(66) substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr(33), Arg(66), and Trp(106) prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp(106) but are likely to include Asp(38) and Cys(39).