Involvement of the Flavin si-Face Tyrosine on the
Structure and Function of Ferredoxin-NADP+ Reductases*
Adrián K.
Arakaki
,
Elena G.
Orellano§,
Nora B.
Calcaterra§,
Jorgelina
Ottado§, and
Eduardo A.
Ceccarelli§¶
From the Molecular Biology Division, Instituto de Biología
Molecular y Celular de Rosario, Consejo Nacional de Investigaciones
Científicas y Técnicas, Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad Nacional de
Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina
In ferredoxin-NADP+ reductase
(FNR), FAD is bound outside of an anti-parallel 
-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+
*
This work was supported in part by grants from Consejo
Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Fondo para la Investigación Científica y Tecnológica
(Argentina) (to E. A. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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