Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP(+) reductase for optimal NADP(+) binding and electron transfer

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg 100 and Arg264 may be directly involved in the proper NADP(+)/NADPH orientation for an efficient electron-transfer reacti on. Protein engineering on Arg 100 and Arg264 from Anabaena PCC 7119 FNR ha s been carried out to investigate their roles in complex formation and elec tron transfer to NADP(+) and to ferredoxin/flavodoxin. Arg100 has been repl aced with an alanine, which removes the positive charge, the long side chai n, as well as the ability to form hydrogen bonds, while a charge reversal m utation has been made at Arg264 by replacing it with a glutamic acid. Resul ts with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-stat e as well as fast transient kinetic techniques, and the three-dimensional s tructure of Arg264Glu FNR has been solved. The results reported herein reve al important conceptual information about the interaction of FNR with its s ubstrates. A critical role is confirmed for the long, positively charged si de chain of Arg 100. Studies on the Arg264Glu FNR mutant demonstrate that t he Arg264 side chain is not critical for the nicotinamide orientation or fo r nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer wi th its protein partners, ferredoxin and flavodoxin.