Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP(+) reductase for optimal complex formation and electron transfer to ferredoxin

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromat ic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investiga ted further the role of hydrophobic interactions in complex stabilization a nd ET between these proteins by replacing three hydrophobic residues, Leu-7 6, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from N ADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chai ns. In contrast, only subtle changes are observed upon conservative replace ment. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of thes e mutants. The introduction of bulky aromatic side chains appears to produc e rearrangements of the side chains at the FNR/Fd interaction surface. Thus , subtle changes in the hydrophobic patch influence the rates of ET to and hom Fd by altering the binding constants and the FAD redox potentials, indi cating that these residues are especially important in the binding and orie ntation of Fd for efficient ET. These results are consistent with the struc ture reported for the Anabaena FNR.Fd complex.