Catalytic mechanism of hydride transfer between NADP+/H and ferredoxin-NADP+ reductase from Anabaena PCC 7119.
The mechanism of hydride transfer between Anabaena FNR and NADP+/H was analysed using for the first time stopped-flow photodiode array detection and global analysis deconvolution. The results indicated that the initial spectral changes, occurring within the instrumental dead time upon reaction of FNR with NADP+/H, included not only the initial interaction and complex formation, but also the first subsequent steps of the sequential reactions that involve hydride transfer. Two different charge-transfer complexes formed prior and upon hydride transfer, FNRox-NADPH and FNRrd-NADP+. Detectable amounts of FNRox-NADPH were found at equilibrium, but FNRrd-NADP+ accumulated to a small extent and quickly evolved. The spectral properties of both charge-transfer complexes, for the first time in Anabaena FNR, as well as the corresponding inter-conversion hydride transfer rates were obtained. The need of an adequate initial interaction between NADP+/H and FNR, and subsequent conformational changes, was also established by studying the reactions of two FNR mutants.
Tejero
Jesús
J
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, E-50009 Zaragoza, Spain.
Peregrina
José Ramón
JR
Martínez-Júlvez
Marta
M
Gutiérrez
Aldo
A
Gómez-Moreno
Carlos
C
Scrutton
Nigel S
NS
Medina
Milagros
M
D017033:Anabaena / Q000201:enzymology*; D002384:Catalysis; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*
D009249:NADP; D006859:Hydrogen; D005287:Ferredoxin-NADP Reductase