Thermodynamics of ferredoxin binding to cyanobacterial nitrate reductase

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 K