Ferredoxin-NADP(H) reductase (FNR) is a FAD-containing protein that catalyzes the reversible transfer of electrons between NADP(H) and ferredoxin or flavodoxin. This enzyme participates in the redox-based metabolism of plastids, mitochondria, and bacteria. Plastidic plant-type FNRs are very efficient reductases in supporting photosynthesis. They have a strong preference for NADP(H) over NAD(H), consistent with the main physiological role of NADP(+) photoreduction. In contrast, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover rates that are up to 100-fold lower than those of their plastidic and cyanobacterial counterparts. With the aim of elucidating the mechanisms by which plastidic enzymes achieve such high catalytic efficiencies and NADP(H) specificity, we investigated the manner in which the NADP(H) nicotinamide enters and properly binds to the catalytic site. Analyzing the interaction of different nucleotides, substrate analogues, and aromatic compounds with the wild type and the mutant Y308S-FNR from pea, we found that the interaction of the 2'-P-AMP moiety from NADP(+) induces a change that favors the interaction of the nicotinamide, thereby facilitating the catalytic process. Furthermore, the main role of the terminal tyrosine, Y308, is to destabilize the interaction of the nicotinamide with the enzyme, inducing product release and favoring discrimination of the nucleotide substrate. We determined that this function can be replaced by the addition of aromatic compounds that freely diffuse in solution and establish a dynamic equilibrium, reversing the effect of the mutation in the Y308S-FNR mutant.
Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs, EC 1.18.1.2) are a widely distributed class of flavoenzymes that have non-covalently bound FAD cofactor as a redox center. FNRs participate in a wide variety of redox-based metabolic reactions, transferring electrons between obligatory one-and two-electron carriers and therefore functioning as a general electron splitter. In non-phototrophic bacteria and eukaryotes, the reaction is driven towards ferredoxin (Fd) reduction, providing reducing power for multiple metabolic pathways, including steroid hydroxylation in mammalian mitochondria, nitrite reduction and glutamate synthesis in heterotrophic tissues of vascular plants, radical propagation and scavenging in prokaryotes, and hydrogen and nitrogen fixation in anaerobes (for a review, see [1,2]). In plants, FNR participates in photosynthetic electron transport, reducing Fd at the level of photosystem I, and transferring electrons to NADP + . This process ends with the formation of the NADPH necessary for CO 2 fixation and other biosynthetic pathways [2].The three-dimensional structures of several FNRs have been determined. They display similar structural features, which have been defined as the prototype for a large family of flavoenzymes [3][4][5][6][7][8][9][10] Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20-to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.
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