Dynamics of the active site architecture in plant-type ferredoxin-NADP+ reductases catalytic complexes

Abstract

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.

ArabidopsisFNRL protein is an NADPH‐dependent chloroplast oxidoreductase resembling bacterial ferredoxin‐NADP+reductases

“…G). This type of band is attributed to charge‐transfer interactions between the oxidised flavin isoalloxazine ring and the nicotinamide ring of NADPH, FAD ox ‐NADPH charge‐transfer complexes (CTC) (Batie and Kamin , Tejero et al , Lans et al , Sánchez‐Azqueta et al ). Evolution of absorption in both flavin band‐I and CTC bands in the following spectra indicates flavin reduction by HT without the appearance of other CTC bands.…”

“…; Tejero et al , Bortolotti et al , Sánchez‐Azqueta et al , ). Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ). Notably, differences in CTCs stabilisation have also been reported between bacterial‐type FPRs relative to plastidic FNRs, as well as upon mutation of key residues in plastid FNRs (Lans et al , Peregrina et al , Bortolotti et al , Sánchez‐Azqueta et al ).…”

“…FNRL is considerably slower in the HT process from NADPH than the leaf‐type, root‐type or bacterial FNRs (Fig. ; Tejero et al , Bortolotti et al , Sánchez‐Azqueta et al , ). Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ).…”

“…Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ). Notably, differences in CTCs stabilisation have also been reported between bacterial‐type FPRs relative to plastidic FNRs, as well as upon mutation of key residues in plastid FNRs (Lans et al , Peregrina et al , Bortolotti et al , Sánchez‐Azqueta et al ). Such differences are related to dissimilarities in the geometric disposition of the reacting rings in the catalytically competent complexes, in the nature of residues at the re ‐face of the isoalloxazine where the coenzyme nicotinamide moiety is expected to stack for catalysis, or to the bipartite binding of the coenzyme (Sánchez‐Azqueta et al ).…”

Identification of Inhibitors Targeting Ferredoxin-NADP+ Reductase from the Xanthomonas citri subsp. citri Phytopathogenic Bacteria

“…Plant-type FNRs are subdivided into plastidic-type (herein FNR) and bacterial-type (herein FPR and including Xcc FPR). The bacterial-types catalyze the electron transfer from NADPH to different electron transfer proteins and are mainly involved in nitrogen fixation and detoxification (including ROS) processes [ 11 , 12 ]. On the contrary, the main function of plastidic-type FNRs is to catalyze the photosynthetic electron transfer from PSI to NADP + to produce NADPH, but they are also able to efficiently provide electrons from NADPH to several electron carrier proteins [ 7 , 8 , 13 , 14 , 15 ].…”