Modulation of the enzymatic efficiency of ferredoxin‐NADP(H) reductase by the amino acid volume around the catalytic site

Abstract

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.

Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from photosystem I to NADP+

“…FNR is highly specific for NADP + /H versus NAD + /H and different studies have established a role for several FNR residues in determining coenzyme binding, specificity and enzymatic efficiency [114–120]. Three FNR regions appear to be mainly responsible for the interaction: 2′‐phospho‐AMP (2′P‐AMP) and pyrophosphate of the NADP + /H binding sites, and the position occupied by the C‐terminal residue where the nicotinamide portion of NADP + (NMN) is proposed to bind for hydride transfer [114–117,119]. S223 and Y235 at the An FNR 2′P‐AMP site are critical in determining the specificity and efficient coenzyme orientation [99,115,121].…”

“…The FNR protein portion has a dual role in this process by: (a) modulating the FAD midpoint potential to a value that makes the hydride transfer reversible, and (b) providing the environment for an efficient encoun-ter between the N5 of the flavin and the C4 of the nicotinamide. FNR is highly specific for NADP + ⁄ H versus NAD + ⁄ H and different studies have established a role for several FNR residues in determining coenzyme binding, specificity and enzymatic efficiency [114][115][116][117][118][119][120]. Three FNR regions appear to be mainly responsible for the interaction: 2¢-phospho-AMP (2¢P-AMP) and pyrophosphate of the NADP + ⁄ H binding sites, and the position occupied by the C-terminal residue where the nicotinamide portion of NADP + (NMN) is proposed to bind for hydride transfer [114][115][116][117]119].…”

“…S223 and Y235 at the AnFNR 2¢P-AMP site are critical in determining the specificity and efficient coenzyme orientation [99,115,121]. The 155-160 and 261-268 loops, which accommodate the coenzyme pyrophosphate portion, also confer specificity and the volume of residues in the latter loop fine-tunes FNR catalytic efficiency [114,116,119,120]. R100 (K166 in spFNR), situated at the FAD-binding domain, allows its guanidinium group to H-bond to the NADP + pyrophosphate, providing the necessary flexibility to address the NMN moiety of NADP + towards the active site [99,108,114].…”

The Plant–Type Ferredoxin-NADP+ Reductases

“…For unstable FNR mutants, longer induction periods (10-14 h) at low temperature (18-20ºC) are recommended. In these cases, lower inductor concentration (0.1 mM IPTG) gives better yields (Musumeci et al, 2008, Musumeci et al, 2011. Nevertheless, extremely long induction periods (20 h or more) are detrimental for the quality of the obtained FNR due to the incorporation of a modified flavin or an improper protein folding ( Figure 3A).…”

“…Inset: absorbance change at 515 nm plotted as function of NADP + concentration. C) Differential spectra elicited by NADP + binding to different FNR mutants (grey, yellow, cyan, red and blue corresponds to the spectra of wildtype, C266A, C266AL266A and C266M FNR forms respectively (Musumeci et al, 2008)).…”