The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.