Role of the C-terminal tyrosine of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase in the electron transfer processes with its protein partners ferredoxin and flavodoxin.
The catalytic mechanism proposed for ferredoxin-NADP(+) reductase (FNR) is initiated by reduction of its flavin adenine dinucleotide (FAD) cofactor by the obligatory one-electron carriers ferredoxin (Fd) or flavodoxin (Fld) in the presence of oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)). The C-terminal tyrosine of FNR, which stacks onto its flavin ring, modulates the enzyme affinity for NADP(+)/H, being removed from this stacking position during turnover to allow productive docking of the nicotinamide and hydride transfer. Due to its location at the substrate-binding site, this residue might also affect electron transfer between FNR and its protein partners. We therefore studied the interactions and electron-transfer properties of FNR proteins mutated at their C-termini. The results obtained with the homologous reductases from pea and Anabaena PCC7119 indicate that interactions with Fd or Fld are hardly affected by replacement of this tyrosine by tryptophan, phenylalanine, or serine. In contrast, electron exchange is impaired in all mutants, especially in the nonconservative substitutions, without major differences between the eukaryotic and the bacterial FNR. Introduction of a serine residue shifts the flavin reduction potential to less negative values, whereas semiquinone stabilization is severely hampered, introducing further constraints to the one-electron-transfer processes. Thus, the C-terminal tyrosine of FNR plays distinct and complementary roles during the catalytic cycle, (i) by lowering the affinity for NADP(+)/H to levels compatible with steady-state turnover, (ii) by contributing to the flavin semiquinone stabilization required for electron splitting, and (iii) by modulating the rates of electron exchange with the protein partners.
Nogués
Isabel
I
Departamento de Bioquímica y Biología Molecular y Celular and Biocomputation and Complex Systems Physics Institute, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Tejero
Jesús
J
Hurley
John K
JK
Paladini
Darío
D
Frago
Susana
S
Tollin
Gordon
G
Mayhew
Stephen G
SG
Gómez-Moreno
Carlos
C
Ceccarelli
Eduardo A
EA
Carrillo
Néstor
N
Medina
Milagros
M
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004579:Electron Transport / Q000502:physiology*; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D007834:Lasers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D014443:Tyrosine / Q000378:metabolism*
D001426:Bacterial Proteins; D005288:Ferredoxins; D005418:Flavodoxin; D010940:Plant Proteins; D014443:Tyrosine; D005287:Ferredoxin-NADP Reductase