A single in vivo-selected point mutation in the active center ofToxoplasma gondiiferredoxin-NADP+reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

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Abstract
Electron transfer between plant-type [2Fe–2S] ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) depends on the physical interaction between both proteins. We have applied a random mutagenesis approach with subsequent in vivo selection using the yeast two-hybrid system to obtain mutants of Toxoplasma gondii FNR with higher affinity for Fd. One mutant showed a 10-fold enhanced binding using affinity chromatography on immobilized Fd. A single serine-to-arginine exchange in the active site was responsible for its increased affinity. The mutant reductase was also enzymatically inactive. Homology modeling of the mutant FNR–Fd complex predicts substantial alterations of protein–FAD interactions in the active site of the enzyme with subsequent structural changes. Collectively, for the first time a point mutation in this important class of enzymes is described which leads to greatly enhanced affinity for its protein ligand