Ferredoxin-NADP⁺ reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP⁺, which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP⁺ interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP⁺ and showed increased K_m for this nucleotide. The EcFPR activity was inhibited by NADP⁺ but this behavior disappeared as arginines were removed. A NADP⁺ analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP⁺ release. Our results give evidence for a new model of NADP⁺ binding and catalysis in bacterial FNRs.We propose that this tight NADP⁺ binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis.

中文翻译：

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由于特定的 NADP+ 结合模式，细菌铁氧还蛋白-NADP+ 还原酶的新催化机制

铁氧还蛋白-NADP ⁺还原酶 (FNR) 是普遍存在的参与氧化还原代谢的黄素酶。FNR 催化 NADP(H) 与铁氧还蛋白或黄素氧还蛋白之间的可逆电子转移。它们被归类为植物型和线粒体型 FNR。植物型 FNR 分为质体类和细菌类。质体 FNR 的周转数比细菌酶高 20 到 100 倍，这些差异与它们的生理功能有关。我们证明了纯化的大肠杆菌FPR (EcFPR) 含有紧密结合的 NADP ⁺，这在质体型 FNR 中不存在。EcFPR 的三维结构证明了 NADP ⁺与三个精氨酸（R144、R174 和 R184）相互作用，可以产生非常高的亲和力和结构化位点。这些精氨酸在其他细菌 FNR 中是保守的，但在质体酶中不保守。我们用Pisum sativum FNR (PsFNR)中类似位置的残基交叉取代了 EcFPR 精氨酸，并用 PsFNR中的精氨酸替换了这些氨基酸。我们通过结构、动力学和稳定性研究分析了所有蛋白质。我们发现 EcFPR 突变体不含结合的 NADP ⁺并且显示该核苷酸的K _m增加。EcFPR 活性被 NADP ⁺抑制，但这种行为随着精氨酸的去除而消失。一个国家发展计划⁺烟酰胺部分的类似物对 EcFPR 产生激活作用并促进 NADP ⁺释放。我们的研究结果为细菌 FNR中 NADP ⁺结合和催化的新模型提供了证据。我们认为，这种紧密的 NADP ⁺结合构成​​了参与氧化还原稳态的细菌 FNR 的基本催化和调节机制。