Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase

https://doi.org/10.1016/j.bbabio.2010.05.006Get rights and content
Under an Elsevier user license
open archive

Abstract

Two transient charge–transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNRox-NADPH (CTC-1), and FNRrd-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.

Abbreviations

FNR
ferredoxin-NADP+ reductase
FNRox
FNR in the fully oxidized state
FNRrd
FNR in the hydroquinone (fully reduced) state
2′-P
2′-phosphate group of NADP+/H
ET
electron transfer
HT
hydride transfer
WT
wild-type
MC
Michaelis complex
CTC
charge–transfer complex
CTC-1
FNRox-NADPH CTC
CTC-2
FNRrd-NADP+ CTC
2′-P-AMP
2′-P-AMP moiety of NADP+/H
kA  B, kB  C
apparent rate constants obtained by global analysis of spectral kinetic data
kobsHT, kobsHT-1, kobsDT, kobsDT-1
observed conversion HT and DT rate constants for the forward and reverse reactions
kHT, kHT-1
hydride transfer first-order rate constants for the forward and reverse reactions, respectively
kDT, kDT-1
deuteride transfer first-order rate constants for the forward and reverse reactions, respectively
KdNADPH, KdNADP+
dissociation constants for the intermediate complexes in the reduction and reoxidation of FNR, respectively: FNR4, T155G/A160T/L263P/Y303S FNR variant
KIE
kinetic isotopic effect

Keywords

Ferredoxin-NADP+ reductase
Hydride transfer
Charge–transfer complex
Site-directed mutagenesis
Kinetic isotopic effect
Isoalloxazine–nicotinamide interactions

Cited by (0)