Further Characterization by Site-Directed Mutagenesis of the Protein-Protein Interface in the Ferredoxin/Ferredoxin:NADP+ Reductase System from Anabaena: Requirement of a Negative Charge at Position 94 in Ferredoxin for Rapid Electron Transfer

Ferredoxins are small electron transfer proteins found ubiquitously in nature. In green plant photosynthesis, the soluble [2Fe2S] ferredoxin shuttles electrons from Photosystem I to ferredoxin:NADP+ reductase. In order to define the features of the protein/protein interface required for efficient electron transfer from ferredoxin to ferredoxin:NADP+ reductase, we have made site-directed mutants of the ferredoxin from the cyanobacterium Anabaena 7120 and measured the rate constants for electron transfer to ferredoxin:NADP+ reductase using laser flash photolysis. Previous results from this laboratory identified two residues in ferredoxin that were crucial to electron transfer between these proteins. One such position (F65) was subsequently shown to require an aromatic amino acid, and it was further shown that interprotein electron transfer was rate-limiting in the case of the slowly reacting F65A mutant (Hurley et al., 1993 J. Am. Chem. Soc. 115, 11,698-11,701). The second crucial position (E94) is shown in the present study to require a negative charge in order to maintain wild-type-like electron transfer reactivity toward ferredoxin:NADP+ reductase. Further, we also demonstrate, for the slowly reacting E94Q mutant, that electron transfer is the rate-limiting step in the interprotein interaction.