Sánchez-Azqueta Ana, Herguedas Beatriz, Hurtado-Guerrero Ramón, Hervás Manuel, Navarro José A, Martínez-Júlvez Marta, Medina Milagros
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Institute of Biocomputation and Physics of Complex Systems (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain.
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Institute of Biocomputation and Physics of Complex Systems (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain; Fundación ARAID, Gobierno de Aragón, Spain.
Biochim Biophys Acta. 2014 Feb;1837(2):251-63. doi: 10.1016/j.bbabio.2013.10.010. Epub 2013 Nov 4.
Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP(+)) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP(+) in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.
铁氧化还原蛋白 - 烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）还原酶（FNR）催化光合生物中还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的生成。在该过程中，其黄素腺嘌呤二核苷酸（FAD）辅因子分两个连续步骤从两个还原型铁氧化还原蛋白（Fd）分子获取两个电子，并在一个单氢化物转移（HT）步骤中将它们转移至NADP(+)。尽管对这种催化机制已有深入了解，但对于已报道的关键残基仍可设想其具有其他作用，并且在先前未被确定在该机制中起特定作用的残基上发现了新特性。在此，我们首次分析了鱼腥藻FNR中Ser59的作用，最近的理论模拟表明该残基可能通过与Ser80协同作用参与活性位点中辅酶的有效结合。我们发现Ser59间接调节活性位点的几何结构、与底物的相互作用以及异咯嗪环的电子性质，进而影响电子转移（ET）和HT过程。此外，我们重新审视了先前在植物同源酶中研究过的Tyr79和Ser80的作用。我们的结果表明，FNR的活性位点由一个氢键网络调节，该网络涉及这些残基的侧链，并且对于关键的最佳底物结合、电子交换，特别是在可逆HT事件中辅酶烟酰胺部分的C4n（氢化物受体/供体）的有效排列至关重要。
