Comparison of the electrostatic binding sites on the surface of ferredoxin for two ferredoxin-dependent enzymes, ferredoxin-NADP(+) reductase and sulfite reductase.
Plant-type ferredoxin (Fd), a [2Fe-2S] iron-sulfur protein, functions as an one-electron donor to Fd-NADP(+) reductase (FNR) or sulfite reductase (SiR), interacting electrostatically with them. In order to understand the protein-protein interaction between Fd and these two different enzymes, 10 acidic surface residues in maize Fd (isoform III), Asp-27, Glu-30, Asp-58, Asp-61, Asp-66/Asp-67, Glu-71/Glu-72, Asp-85, and Glu-93, were substituted with the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, the redox potential of which was more positive by 67 mV than that of the wild type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interestingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, whereas this relationship was reversed in the reaction with SiR. The static interaction of the mutant Fds with each the two enzymes was analyzed by gel filtration of a mixture of Fd and each enzyme, and by affinity chromatography on Fd-immobilized resins. The contributions of Asp-66/Asp-67 and Glu-93 were found to be most important for the binding to FNR and SiR, respectively, in accordance with the kinetic data. These results allowed us to map the acidic regions of Fd required for electron transfer and for binding to FNR and SiR and demonstrate that the interaction sites for the two enzymes are at least partly distinct.
Akashi
T
T
Division of Enzymology, Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.
Matsumura
T
T
Ideguchi
T
T
Iwakiri
K
K
Kawakatsu
T
T
Taniguchi
I
I
Hase
T
T
D029681:Arabidopsis Proteins; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D055672:Static Electricity; D050889:Sulfite Reductase (Ferredoxin); D003313:Zea mays
D029681:Arabidopsis Proteins; D005288:Ferredoxins; D010940:Plant Proteins; D005287:Ferredoxin-NADP Reductase; D050862:Oxidoreductases Acting on Sulfur Group Donors; C488954:SIR protein, Arabidopsis; D050889:Sulfite Reductase (Ferredoxin)