Lys75 of Anabaena Ferredoxin-NADP+ Reductase Is a Critical Residue for Binding Ferredoxin and Flavodoxin during Electron Transfer

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• 作者：Marta Martí ; nez-Jú ; lvez ; Milagros Medina ; John K. Hurley ; Rameh Hafezi ; Tammy B. Brodie ; Gordon Tollin ; and Carlos G&oacute ; mez-Moreno
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 29, 1998
• 年：1998
• 卷：37
• 期：39
• 页码：13604 - 13613
• 全文大小：115K
• 年卷期：v.37,no.39(September 29, 1998)
• ISSN：1520-4995

文摘

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP⁺ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in theinteraction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess thispossibility, this residue has been replaced by another positively charged residue, Arg, by two unchargedresidues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, andCD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate thatall the mutated proteins folded properly and that significant protein structural rearrangements did notoccur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP,indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) betweenFNR and NADP⁺ or NADPH. However, steady-state kinetic activities requiring complex formation andET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75was removed, and the ET reaction was not even measurable if a negatively charged residue was placedat this position. These kinetic parameters also suggest that it is complex formation that is affected bymutation. Consistent with this, when dissociation constants (K_d) for FNR_ox-Fd_ox (differential spectroscopy)and FNR_ox-Fd_rd (laser flash photolysis) were measured, it was found that neutralization of the positivecharge at position 75 increased the K_d values by 50-100-fold, and that no complex formation could bedetected upon introduction of a negative charge at this position. Fast transient kinetic studies alsocorroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects thecomplex formation process with its protein partners but indicates that ET is still achieved in all the reactions.This study thus clearly establishes the requirement of a positive charge at position Lys75 for complexformation during ET between FNR and its physiological protein partners. The results also suggest thatthe interaction of this residue with its protein partners is not structurally specific, since Lys75 can still beefficiently substituted by an arginine, but is definitely charge specific.