AN AROMATIC AMINO-ACID IS REQUIRED AT POSITION-65 IN ANABAENA FERREDOXIN FOR RAPID ELECTRON-TRANSFER TO FERREDOXIN NADP+ REDUCTASE

Vegetative cell ferredoxin (Fd) from the cyanobacterium Anabaena 7120 functions in photosynthesis to transfer electrons from photosystem I to ferredoxin:NADP+ reductase (FNR). Previous work using site-directed mutagenesis and laser flash photolysis (Hurley et al., Biochemistry 1993, 32, 9346-9354) has shown that replacing Phe-65 with aliphatic amino acids decreased the rate constant for the Fd to FNR electron-transfer (ET) reaction by more than 3 orders of magnitude, while leaving other properties of the protein (UV-vis and CD spectra and reduction potential, etc.) intact. The present study demonstrates that replacing Phe-65 with either of the aromatic residues Trp or Tyr restores wild type (wt) activity with regard to ET to FNR. These mutants also restored wt ionic strength dependencies of the Fd to FNR ET rate constant, which, for the aliphatic Phe-65 mutants, were vastly different from those of the wt. In addition, a double mutant which places an aliphatic residue at position 65 and an aromatic residue at an adjacent site also reacted more than 4 orders of magnitude slower in ET to FNR. The observed rate constant for ET to FNR of the Phe-65 to Ala-65 mutant was found to be independent of FNR concentration, indicating that this rate constant reflects processes occurring during intracomplex ET between the two proteins. These results clearly demonstrate that Fd from Anabaena 7120 requires an aromatic amino acid at position 65 for efficient ET to FNR.