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The role of the seven negatively charged amino acids of Synechocystis sp. PCC 6803 ferredoxin (Fd), i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92, and Glu93, predicted to form complex with nitrate reductase (NR), was investigated using site-directed mutagenesis and isothermal titration calorimetry (ITC). These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92, and Glu93, that are essential for the Fd binding and efficient electron transfer to the NR. ITC measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7 μM for the complex at low ionic strength residues and both the enthalpic and entropic components are associated with complex formation. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q, and E93N demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively charged Fd residues at these positions are important for the effective and productive interaction with wild-type enzyme.