Roles of negatively charged surface residues of putidaredoxin in interactions with redox partners in p450cam monooxygenase system.
To investigate the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam system, we focused on the role of negatively charged surface amino acid residues. The amino acid residues we examined in this mutational study are Asp-58, Glu-65, Glu-72, and Glu-77, which are located on the alpha-helical segment to form a negatively charged region on the surface of Pdx and have been supposed to play key roles in the association with the redox partners, NADH-putidaredoxin reductase (PdR) and P450cam. The neutralization of the single negative charge on these amino acid residues did not significantly inhibit the electron-transfer reaction with the redox partners, except for the mutation at Glu-72. Together with the previous results, we can conclude that the negatively charged cluster on the alpha-helical segment is not so crucial for the electron transfer of the Pdx/PdR complex, and, instead of the negative charges, the steric hindrance is essential for the binding of Pdx with PdR. In the electron transfer from Pdx to P450cam, the alpha-helical region would not be included in the binding site with P450cam and some specific hydrogen bonds on the surface loop near the Fe-S center contribute to the electron transfer to P450cam. Such different binding sites and interactions for Pdx will shed light on the electron-transfer mechanism mediated by Pdx, the shuttle mechanism.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
M
M
Aoki
K
K
Ishimori
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I
Morishima
D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019475:Camphor 5-Monooxygenase / Q000378:metabolism*; D004560:Electricity; D004579:Electron Transport; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011199:Potentiometry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013053:Spectrophotometry
D005288:Ferredoxins; D009243:NAD; D001224:Aspartic Acid; D018698:Glutamic Acid; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase