C-Terminal Tyrosine of Ferredoxin-NADP+ Reductase in Hydride Transfer Processes with NAD(P)+/H

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• 作者：Jesú ; s Tejero ; Inmaculada Pé ; rez-Dorado ; Celia Maya ; Marta Martí ; nez-Jú ; lvez ; Julia Sanz-Aparicio ; Carlos Gó ; mez-Moreno ; Juan A. Hermoso ; and Milagros Medina
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：October 18, 2005
• 年：2005
• 卷：44
• 期：41
• 页码：13477 - 13490
• 全文大小：1004K
• 年卷期：v.44,no.41(October 18, 2005)
• ISSN：1520-4995

文摘

Ferredoxin-NADP⁺ reductase (FNR) catalyzes the reduction of NADP⁺ to NADPH in anoverall reversible reaction, showing some differences in the mechanisms between cyanobacterial and higherplant FNRs. During hydride transfer it is proposed that the FNR C-terminal Tyr is displaced by thenicotinamide. Thus, this C-terminal Tyr might be involved not only in modulating the flavin redoxproperties, as already shown, but also in nicotinamide binding and hydride transfer. FNR variants fromthe cyanobacterium Anabaena in which the C-terminal Tyr has been replaced by Trp, Phe, or Ser havebeen produced. All FNR variants show enhanced NADP⁺ and NAD⁺ binding, especially Tyr303Ser,which correlates with a noticeable improvement of NADH-dependent reactions. Nevertheless, the Tyr303Servariant shows a decrease in the steady-state k_cat value with NADPH. Fast kinetic analysis of the hydridetransfer shows that the low efficiency observed for this mutant FNR under steady-state conditions is notdue to a lack of catalytic ability but rather to the strong enzyme-coenzyme interaction. Three-dimensionalstructures for Tyr303Ser and Tyr303Trp variants and its complexes with NADP⁺ show significantdifferences between plant and cyanobacterial FNRs. Our results suggest that modulation of coenzymeaffinity is highly influenced by the strength of the C-terminus-FAD interaction and that subtle changesbetween plant and cyanobacterial structures are able to modify the energy of that interaction. Additionally,it is shown that the C-terminal Tyr of FNR lowers the affinity for NADP⁺/H to levels compatible withsteady-state turnover during the catalytic cycle, but it is not involved in the hydride transfer itself.