2011-12-30T23:02:05Z

Yoann Styczen: /* Enzyme */

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 and chemical modification and site-directed mutagenesis experiments give features of the structure that are important for function.
{{STRUCTURE_1frn| PDB=1frn}}
== Description ==
=== Enzyme ===
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+.

Ferredoxin-NADP+ reductase owns two different substrates :
*reduced ferredoxin
*NADP+

This protein also have one cofactor :
*FAD

FNR catalyzes the following reaction :

2Fdreduced + NADP+ + H+ → 2Fdoxidized + NADPH

=== Activity ===

== Structure ==
<Structure load='1frn' size='500' frame='true' align='right' caption='T5 5'exonuclease' />
=== General 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 <ref name = Karplus1>PMID:7897656</ref>.

Ferredoxin-NADP+ reductase of spinach is made of two structural domains :
*The <scene name='User:Yoann_Styczen/Sandbox_203/Domain1/1'>first domain</scene>, from the residue 20 to 161 at the N-ter of the protein, is a six-stranded antiparallel β-barrel with a peripheral two-stranded antiparallel hairpin and a final α-helix of eight residues <ref name=Karplus2>PMID:8027025</ref>. This domain contain the binding site for the FAD cofactor <ref name=Aliverti>doi:10.1016/j.abb.2008.02.014</ref> which binds its adenosine-diphosphate part to the hairpin and helix <ref name=Karplus2>PMID:8027025</ref>. <scene name='User:Yoann_Styczen/Sandbox_203/Interface/1'>Residues 20 to 27 and 150 to 161</scene> are involved in the interface between FAD-binding domain and the second domain of the flavoprotein <ref name=Karplus2>PMID:8027025</ref>.
*The <scene name='User:Yoann_Styczen/Sandbox_203/Domain2/1'>second domain</scene>, from the residue 162 to 314 at the C-ter of the protein, contains a central five-stranded parallel β-sheet and six associated α-helix <ref name=Karplus2>PMID:8027025</ref>. This terminal domain is where the NADP+ binds <ref name=Paladini>doi:10.1021/bi9004232</ref>.

The interface between these two domains contains the active site of the ferredoxin-NADP+ reductase and the large shallow cleft between the two domains provides for ferredoxin a cavity, close to the protein surface and distant from the active site.
This cleft is maybe involved in membrane atachment <ref name = Karplus1>PMID:7897656</ref>.

Moreover the <scene name='User:Yoann_Styczen/Sandbox_203/Cysres/3'>five cystein residues</scene> (Cys42, Cys114, Cys132, Cys137 and Cys272) are present as sulfhydryls and not disulfide bridges <ref name=Karplus2>PMID:8027025</ref>. This precision is important for two of these (<scene name='User:Yoann_Styczen/Sandbox_203/2cysres/2'>Cys132 and Cys272</scene>) since they are implicated in catalytic activity of the enzyme.

=== Residues involved in binding of substrates and cofactors ===
==== NADP 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 <ref name=Karplus3>PMID:1986412</ref> but the fixation of the nicotinamide portion is still not well known.
The 2'-phospho-AMP binds at the C-ter end of the <scene name='User:Yoann_Styczen/Sandbox_203/Nadpbetasheet/1'>β-sheet</scene> in NADP+-binding domain : the adenine is fixed between <scene name='User:Yoann_Styczen/Sandbox_203/Adenine/1'>Tyr246 and Leu274</scene> and the 2'-phosphate is recognized by <scene name='User:Yoann_Styczen/Sandbox_203/Othernadpportionfixation/1'>Ser234, Arg235, Tyr246 and probably the Lys244 residue</scene> <ref name=Karplus2>PMID:8027025</ref>. Indeed, mutation of <scene name='User:Yoann_Styczen/Sandbox_203/Lys244/1'>Lys244</scene> 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 <ref name=Aliverti2>PMID:1917920</ref>.

The <scene name='User:Yoann_Styczen/Sandbox_203/Lys116/1'>Lys116</scene> 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 <ref name=Karplus2>PMID:8027025</ref>. 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 <ref name=Aliverti2>PMID:1917920</ref>.

About the nicotinamide portion of NADP+, it is supposed that the fixation involves a conformational change in FNR with the deplacement of <scene name='User:Yoann_Styczen/Sandbox_203/Tyr314/1'>Tyr314</scene>. 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 <ref name=Karplus3>PMID:1986412</ref>.

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

==== Ferredoxin binding ====
==== FAD binding ====
The cofactor of FNR, the FAD, is tightly bound outside of the antiparallel β-barrel, 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 Tyr95, Ser96 and Tyr314, which interact with the frond and back sides of the flavin (Tyr314 actually belongs to the NADP+ binding domain, but folds back into the FAD binding site). The pyrophosphate is fixed by Arg93 and peptide amides from Gly130 to Ser133 of the first turn of the α-helix. Ser133 assure FAD binding by making hydrogen-bond to the phosphoryl group <ref name=Karplus2>PMID:8027025</ref>.

Mutagenesis studies of cysteine residues <ref name=Aliverti3>PMID:8518283</ref> showed that the well conserved Cys272 is possibly involved in FAD binding or catalytic activity.

=== Important residues for structure and catalytic activity ===
==== Structure ====
==== Catalytic activty ====
As seen before, the phenol side chain of the Tyr314 is displaced when the nicotinamide binds to FNR and two other residues, Ser96 and Cys272, interact respectively with the N5 of FAD and C4 of NADP+, which are the two atoms involved in hybrid transfer <ref name=Karplus4>PMID:8298460</ref>.
Indeed mutagenesis assays showed that mutation of Ser96 resulted in a big decrease of the FNR catalytic activity by disturbing interaction between isoalloxazine ring of FAD and nicotinamide ring of NADP+ <ref name=Aliverti4>PMID:7677850</ref>. But mutation of Cys272 only caused a moderate decline of the catalytic activity <ref name=Aliverti3>PMID:8518283</ref>.

Several possibilities are suggested to explain the catalytic role of these residues <ref name=Karplus2>PMID:8027025</ref> :
#The Ser96 peptide hydrogen bond to N5 could lead to push out the N5 proton present in reduced state of the flavin plane towards nicotinamide.
#The Ser96 hydroxyl could accept a hydrogen bond to stabilize the reduced flavin and it could operate during the transition state of hibrid transfer
#Cys272 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.

== References ==
<references />