Role of Arg100 and Arg264 from Anabaena PCC 7119 Ferredoxin-NADP+ Reductase for Optimal NADP+ Binding and Electron Transfer

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• 作者：Marta Martí ; nez-Jú ; lvez ; Juan Hermoso ; John K. Hurley ; Tomá ; s Mayoral ; Julia Sanz-Aparicio ; Gordon Tollin ; Carlos Gó ; mez-Moreno ; and Milagros Medina
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：December 22, 1998
• 年：1998
• 卷：37
• 期：51
• 页码：17680 - 17691
• 全文大小：178K
• 年卷期：v.37,no.51(December 22, 1998)
• ISSN：1520-4995

文摘

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the sidechains of Arg100 and Arg264 may be directly involved in the proper NADP⁺/NADPH orientation for anefficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP⁺and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positivecharge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutationhas been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniquesindicate that the mutated proteins folded properly and that significant protein structural rearrangementsdid not occur. Both mutants have been kinetically characterized by steady-state as well as fast transientkinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The resultsreported herein reveal important conceptual information about the interaction of FNR with its substrates.A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264GluFNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or fornicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behaviorin its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.