Comparison of the electrostatic binding sites on the surface of ferredoxinfor 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 prot ein-protein interaction between Fd and these two different enzymes, 10 acid ic 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 wit h the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, t he redox potential of which was more positive by 67 mV than that of the wil d type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interes tingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, wh ereas this relationship was reversed in the reaction with SiR. The static i nteraction 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 chromatograp hy 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, respective ly, in accordance with the kinetic data. These results allowed us to map th e acidic regions of Fd required for electron transfer and for binding to FN R and SiR and demonstrate that the interaction sites for the two enzymes ar e at least partly distinct.