Abstract
In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)
Highlights
Ferredoxin-NADPϩ reductases (FNR,1 1.18.1.2) participate in a broad range of redox metabolic pathways in a wide variety of organisms [1, 2]
In ferredoxin-NADP؉ reductase (FNR), FAD is bound outside of an anti-parallel -barrel with the isoalloxazine lying in a two-tyrosine pocket
We observed that in FNR-like flavoproteins the tyrosine near the si-face of the flavin is completely conserved and always interacting in an edge-to-face position (Table I, Fig. 1 for pea FNR), which has been proved to be a favorable orientation among interacting aromatic molecules [39]
Summary
Ferredoxin-NADPϩ reductases (FNR,1 1.18.1.2) participate in a broad range of redox metabolic pathways in a wide variety of organisms [1, 2]. We have succeeded in obtaining the structures of productive NADPϩ and NADPH complexes with pea FNR [10] This structure reveals the productive binding mode of NADPϩ to the catalytic site, in which the nicotinamide ring lies against the re-face of the isoalloxazine ring in an angle of ϳ30°. The aromatic moiety of the tyrosine interacts through - stacking directly with the isoalloxazine ring, the hydroxyl group makes a hydrogen bond with the 4Ј-ribityl hydroxyl group of FAD. The latter hydroxyl group is in contact with Lys-110 through a water molecule (H2O 1003). Previous studies have replaced the homologous Tyr-95 of spinach FNR by phenylalanine, observing changes essentially in kcat values of the mutant enzyme respect to the wild type form [25]
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