Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases.
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(+)
Arakaki
A K
AK
Molecular Biology Division, Instituto de Biologia Molecular y Celular de Rosario, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina.
Orellano
E G
EG
Calcaterra
N B
NB
Ottado
J
J
Ceccarelli
E A
EA
D000818:Animals; D001665:Binding Sites; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D019800:Phenol / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism*
D018076:DNA, Complementary; D019800:Phenol; D014443:Tyrosine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D010100:Oxygen