FNR

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Introduction

Ferredoxin-NADP⁺ oxidoreductase (FNR; EC 1.18.1.2) is an enzyme that is ubiquitous in various organisms, including heterotrophic and phototrophic bacteria, in mitochondria and plastids of higher plants and algae, as well as apicoplasts of some intracellular parasites and so on. FNR contains one molecule non-covalently bound FAD as a prosthetic group and it catalyzes the reversible electron transfer between ferredoxin (Fd) (or flavodoxin) and NAD(P)H. The widely studied reaction catalyzed by FNR is the last step in the linear electron transfer chain in chloroplasts. The FAD cofactor of FNR acts as a one-to-two electronic switch by reducing FAD to a semiquinone form FADH. Then there is another round of reduction to FADH−, and the hydride transfer from FADH− to NADP⁺. NADPH is mainly used for CO2 fixation in the Calvin-Benson cycle. In addition to chloroplast FNR, FNR protein also exists in non-photosynthetic plastids of higher plants.

Figure 1. FNR functions in the crossing of electron transfer pathway (Mulo, P. 2011)

Structure of FNR

The chloroplast FNR protein is a hydrophilic protein with a molecular weight of approximately 35 kDa. The sequence similarity of FNR from different plant species ranges from 40% to 97%, especially the regions involved in the binding of FAD and NADP⁺ show a high degree of identity. The three-dimensional structure of chloroplast FNR has been described in many species, and the best resolution is 1.7 Å. The study found that the topology of all chloroplast FNRs is highly conserved, because FNR proteins in all studied species are composed of two different domains, which are connected by a loop. The N-terminal domain (about 150 amino acids) is involved in FAD binding, while the C-terminal domain (about 150 amino acids) is mainly responsible for NADP⁺ binding. FAD-binding domain consists of a β-barrel structure consisting of 6 anti-parallel β strands, and is rcrcovered by an α helix and a long loop. The NADP⁺-binding domain consists of a central five-strand parallel β sheet, surrounded by six α-helices. Among them, the NADP⁺- binding domain can be further divided into two sub-domains. The C-terminal subdomain displays a dynamic conformation at stromal daytime pH, allowing increased binding of NADP⁺, thereby effective functional control for photosynthesis according to ambient illumination.

Figure 2. Structure of FNR in complex with Fd (Mulo, P. 2011)

Function of FNR

• FNR and circulating electron flow around PSI

In addition to the obvious role of FNR in the linear electron transfer reaction of photosynthesis, many studies have shown that FNR also participates in cyclic electron flow around PSI. In cyclic electron flow, electrons are transferred from the PSI to the Cyt b6f complex through Fd, and a proton gradient is formed at the same time. Therefore, cyclic electron transfer produces ATP without causing the accumulation of NADPH.

It is generally believed that cyclic electron transfer provides the ATP needed for driving the CO2 concentrating mechanism in the C₄ plants. Recent studies have also proved the importance of cyclic electron flow in C₃ plants. There are currently at least two different cyclic electron transfer pathways: 1) In the Fd- or ferredoxin-plastoquinone oxidoreductase (FQR)-dependent pathway, electrons go from the Fd to Cyt b6f complex and return to PSI; 2) In the NDH-dependent pathway, FNR oxidizes Fd to produce NADPH, which may be reduced by the thylakoid-bound NDH complexes. The NDH complex can provide reducing power for the reduction of the plastoquinone pool.

• FNR and oxidative stress

In higher plant chloroplasts PSII and PSI are usually potential sources of harmful reactive oxygen species (ROS) in plant tissues. In E. coli, FNR is involved in the quenching of ROS. Compared with WT CC-125, the steady-state level of chloroplast FNR transcripts showed an increase. In addition, studies have also shown that the expression of plant FNR can restore the oxidative tolerance of mutant E. coli. These results all promote the study of FNR's involvement in the oxidative stress response of higher plants. The propagation of superoxide and methyl viologen, and the subsequent accumulation of H₂O₂ in wheat plants indicate that, compared with bacterial cells, the content of FNR mRNA and protein in higher plants decreases rather than increases when oxidative stress is induced. However, the production of ROS leads to a significant release of FNR from the thylakoid membrane, and subsequent reduction in NADP⁺ photoreduction capacity, which may be aimed at maintaining the NADP⁺/NADPH homeostasis of the stressed plants.

Figure 3. FNR functions in the crossing of electron transfer pathway (Mulo, P. 2011)