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spinqualitylabelsall models PDB ID 1frn Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
1frn, resolution 2.00Å ()
Ligands:	, ,
Activity:	Ferredoxin--NADP(+) reductase, with EC number 1.18.1.2
Structural annotation: Resources: CATH : 1Frn001, 1Frn002 InterPro : Ipr015701, Ipr001709, Ipr001433, Ipr012146 Pfam : PF00175 SCOP : d1frn_2, d1frn_1 UniProt : P00455
Functional annotation: Cellular component: thylakoid chloroplast thylakoid membrane membrane Biological process: photosynthesis transport electron transport Molecular function: NADP binding oxidoreductase activity protein binding ferredoxin-NADP+ reductase activity FAD binding
Evolutionary conservation: Toggle Conservation Colors: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

Ferredoxin-NADP+ reductase is an FAD-containing enzyme that catalyzes the reversible electron transfer between NADP+ and ferredoxin or flavodoxin.

The structure of this enzyme, chemical modifications and site-directed mutagenesis experiments give features about structure that are important for function.

DescriptionDescription

EnzymeEnzyme

Ferredoxin-NADP+ reductase (FNR), also called ferredoxin-NADP+ oxidoreductase, is a flavoprotein which can be mainly found in chloroplasts, mitochondria and bacteria. This enzyme is a member of the familiy of flavoenzymes and also of the family of oxidoreductases, that use iron-sulfur proteins as electron donors and NAD+ or NADP+ as electron acceptors. In plants, this is an ubiquitous hydrophilic protein of about 35 kDa which binds to the stromal surface of the thylakoid membrane.

This protein is involved in the last step of the photosynthetic electron transport chain. Indeed, ferredoxin-NADP+ reductase catalyzes the electron transfer between the electron carrier protein ferredoxin (Fd) and NADP+ to produce NADPH.

Ferredoxin-NADP+ reductase owns two different substrates :

• reduced ferredoxin
• NADP+

This protein also have one cofactor :

• FAD

FNR catalyzes the following reaction :

2Fd_reduced + NADP⁺ + H⁺ → 2Fd_oxidized + NADPH

ActivityActivity

Catalytic pathway of the Ferredoxin-NAPD+ reductase (click to enlarge)

Studies have demonstrated that ferredoxin-NADP+ reductase proceeds catalyse reaction by forming a ternary complex with NADP+ and reduced ferredoxin.

The catalysis takes place in nine steps  :

1. Fixation of NADP+ to the enzyme. If the ferredoxin binds before NADP+, it impair the reaction. This is a mechanism used by oxidized ferredoxin to down-regulate the reaction.
2. Reduced ferredoxin binds to its binding site
3. Ferredoxin transfers very rapidly its electron to the FNR through a one-electron reduction of the flavin of FAD. FNR becomes a semiquinone FNR.
4. NADP+ facilitate the dissociation of oxidized ferredoxin, which are the rate-limiting steps of the reaction.
5. A second reduced ferredoxin binds to the semiquinone FNR
6. Its electron is tranfered to the FNR through a second one-electron reduction of the flavin. Semiquinone FNR becomes a reduced FNR.
7. A hybrid transfer, involving one proton is done to produce NADPH
8. Dissociation of oxidized ferredoxin
9. Dissociation of NADPH

The only differences between reduced FNR and oxidized FNR is an presence of a water molecule at N1 of the flavin and a movement of Ser96 toward N5 atom of the flavin .

StructureStructure

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General 3D structure descriptionGeneral 3D structure description

Structural informations of ferredoxin-NADP+ reductase has been found by crystallography. The most recent crystal structure was reported in the literature at 1.7Å resolution .

Ferredoxin-NADP+ reductase of spinach is made of two structural domains :

• The , from the residue 20 to 161 at the N-ter of the protein, is a six-stranded antiparallel with a peripheral

and a final of eight residues . This domain contain the binding site for the FAD cofactor which binds its adenosine-diphosphate part to the hairpin and helix . are involved in the interface between FAD-binding domain and the second domain of the flavoprotein .

• The , from the residue 162 to 314 at the C-ter of the protein, contains a central and . This domain is where the NADP+ binds .

The interface between these two domains contains the active site of the ferredoxin-NADP+ reductase and the shallow cleft between these two domains provides a cavity for ferredoxin, close to the protein surface and distant from the active site. This cleft is maybe involved in membrane attachment .

Moreover the (Cys42, Cys114, Cys132, Cys137 and Cys272) are present as sulfhydryls and not disulfide bridges . This precision is important for two of these () since they are implicated in catalytic activity of the enzyme.

Residues involved in binding of substrates and cofactorsResidues involved in binding of substrates and cofactors

NADP bindingNADP binding

In order to allow the hybride electron transfer, NADP+ must bind to ferredoxin-NADP+ reductase with its nicotinamide ring close to the flavin. Crystallography has shown how the 2'-phospho-AMP half of NADP+ binds to the enzyme but the fixation of the nicotinamide portion is still not well known. The 2'-phospho-AMP binds at the C-ter end of the in NADP+-binding domain : the adenine is fixed between and the 2'-phosphate is recognized by . Indeed, mutation of with a Gln affect only the dissociation constant for NADPH from the Michaelis complex. So Lys244 help to stabilize the Michaelis complex, by interacting with the adenine part of NADP+ due to its proximity to this part .

The of the FAD domain is also implicated in the interaction with a phosphate groupe of NADP+ near the first α-helix of the NADP+-binding domain . Indeed, Lys116 extends from the FAD domain to make a hydrogen bond to the 5'-phosphate of NADP+ and site-directed mutagenesis confirm its role in NADP binding and nicotinamide placement .

About the nicotinamide portion of NADP+, it is supposed that the fixation involves a conformational change in FNR with the deplacement of phenol group. Tyr314 occupies the nicotinamide binding site in the unliganted structure, and its displacement by the nicotinamide may play a more active role in catalysis by orientating and fixing the nicotinamide in its proper position .

All of the residues mentionned before are completely conserved among the ferredoxin-NADP+ reductase family.

Ferredoxin bindingFerredoxin binding

Currently, no cristallography structure has been determined for the FNR/Ferredoxin complex but there are some biochemical data that help to determine the residues involved in ferredoxin binding. It is also supposed that the ferredoxin binds to the FNR with its iron-sulfur cluster very close to the C7a and the C8a methyl groups of the flavin . This provides clues about ferredoxin orientation.

It is also known that the ionic strength can impact the binding of ferredoxin in FNR. This suggests that charged residues are involved in formation of FNR/ferredoxin complex. Indeed the amino acide sequence of ferredoxin contains a large number of conserved acidic residues which form negative groups interacting with positive groups of FNR, which have been localized by electrostatics calculation. All of implicated residues of FNR are found in the large shallow cleft which contains the exposed part of flavin (with C7a and C8a methyl groups) . Some chemical modifications studies (mainly with lysines) have been done and reveal that four lysine residues of FNR are protected from modification by the binding of ferredoxin . These residues are Lys18, , and and they are, for most them, well conserved.

The involvement of and has been confirmed through experiments with truncated enzyme : when the enzyme starts at residue 33 or 36, she is normally folded (diaphorase activity is conserved) but totally inactive .

FAD bindingFAD binding

The cofactor of FNR, the FAD, is tightly bound outside of the antiparallel , which is the core of the FAD binding domain in the first domain of FNR. The fixation is made deeply in a pocket formed by strand 4 and 5 of the β-barrel. Some ponctual residues help the FAD binding, such , which interact with the front and back sides of the flavin ( actually belongs to the NADP+ binding domain, but folds back into the FAD binding site). The pyrophosphate is fixed by and peptide amides from of the first turn of the α-helix. assure FAD binding by making hydrogen-bond to the phosphoryl group .

Mutagenesis studies of showed that the well conserved is possibly involved in FAD binding or catalytic activity.

Important residues for structure and catalytic activityImportant residues for structure and catalytic activity

StructureStructure

Most of the residue involved in the structure of the protein are also residue involved in the binding of substrates and in the catalytic activity. They are for most of them very conserved and buried in the hydrophobic core of FNR. They can be find in a cluster in the β-barrel and in an other cluster in the parallel β-sheet, so in the two domains of the FNR, suggesting that their folding may be a bit dependent from each other .

The and some other residues (like glycines adopting special dihedral angles, serine and aspartic acid doing hydrogen bonds inside the main chain) which stabilize turn conformations are important for structural integrity .

Moreover it has been demonstrated that substitution of the Tyr308 of the pea FNR (corresponding to the in spinach FNR) destabilize the conformation of the protein . Since we now that the presence of this tyrosine is important for FAD binding, this suggests that FAD attachment is probably involved in FRN structure as a chaperone .

Mutations of with hydrophilic residues impair FRN folding too, since the environnement of Cys42 is very hydrophobic .

Catalytic activtyCatalytic activty

As seen before, the phenol side chain of the is displaced when the nicotinamide binds to FNR and two other residues, and , interact respectively with the N5 of FAD and C4 of NADP+, which are the two atoms involved in hybrid transfer . Indeed mutagenesis assays showed that mutation of resulted in a big decrease of the FNR catalytic activity by disturbing interaction between isoalloxazine ring of FAD and nicotinamide ring of NADP+ . But mutation of only caused a moderate decline of the catalytic activity .

Several possibilities are suggested to explain the catalytic role of these residues  :

1. The peptide hydrogen bond to N5 could lead to push out the N5 proton, present in reduced state, of the flavin plane towards nicotinamide. It is supposed that the N5 proton could have different sources : buried water molecule close to N5 or a proton from the surface-exposed side chain transfered to Ser96 .
2. The hydroxyl could accept a hydrogen bond to stabilize the reduced flavin and it could operate during the transition state of hibrid transfer.
3. residue could help the positioning of the nicotinamide and the sulfur could accept a hydrogen bond from the oxidized nicotinamide in order to facilitate hybrid transfer. Cys272 plays more a supporting role than a crucial role in catalysis.