A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.
The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.
Deng
Z
Z
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Aliverti
A
A
Zanetti
G
G
Arakaki
A K
AK
Ottado
J
J
Orellano
E G
EG
Calcaterra
N B
NB
Ceccarelli
E A
EA
Carrillo
N
N
Karplus
P A
PA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013057:Spectrum Analysis; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics
D008024:Ligands; D014443:Tyrosine; D009249:NADP; D005287:Ferredoxin-NADP Reductase