Literature summary for 1.18.1.2 extracted from

Demmer, J.K.; Huang, H.; Wang, S.; Demmer, U.; Thauer, R.K.; Ermler, U.
Insights into flavin-based electron bifurcation via the NADH-dependent reduced ferredoxin NADP oxidoreductase structure (2015), J. Biol. Chem., 290, 21985-21995 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of the NfnAB complex of Thermotoga maritima in Escherichia coli strain under aerobic conditions	Thermotoga maritima

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant NfnAB free or in complex with NADH, sitting drop vapour diffusion method, 17.5 mg/ml NfnAB protein in 10 mM MOPS-KOH, pH 7.0, 2 mM DTT, and 0.01 mM FAD, room temperature, for the complex soaking the crystals with 5mM NADH for 40 min, multiple wavelength anomalous dispersion X-ray diffraction structure determinatin and analysis at 2.3-2.4 A resolution, modeling	Thermotoga maritima

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kinetic evidence for its bifurcating behavior of the enzyme, cofactor kinetics, overview	Thermotoga maritima

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Thermotoga maritima	5737	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	in iron-sulfur clusters	Thermotoga maritima

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 oxidized ferredoxin + NADPH	Thermotoga maritima	-	2 reduced ferredoxin + NADP+ + H+	-	r

Organism

Organism	UniProt	Comment	Textmining
Thermotoga maritima	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant NfnAB complex of Thermotoga maritima from Escherichia coli strain under aerobic conditions by ultracentrifugation, and heat treatment for 30 min at 80°C, the supernatant is further purified by affinity chromatography and ultrafiltration. Iron-sulfur cluster reconstitution in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions	Thermotoga maritima

Purification (Comment)

Organism

recombinant NfnAB complex of Thermotoga maritima from Escherichia coli strain under aerobic conditions by ultracentrifugation, and heat treatment for 30 min at 80°C, the supernatant is further purified by affinity chromatography and ultrafiltration. Iron-sulfur cluster reconstitution in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions

Thermotoga maritima

Reaction

Reaction	Comment	Organism	Reaction ID
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH	mechanism of FAD-based electron bifurcation, overview	Thermotoga maritima	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 oxidized ferredoxin + NADPH	-	Thermotoga maritima	2 reduced ferredoxin + NADP+ + H+	-	r
2 oxidized ferredoxin + NADPH	complete reversibility of the reaction	Thermotoga maritima	2 reduced ferredoxin + NADP+ + H+	-	r
additional information	the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview. NADH-dependent NADP+ reduction with reduced ferredoxin with a regenerating system composed of Fdox, ferredoxin-dependent [FeFe]-hydrogenase, and 100% H2 as a gas phase. The apparent Km value of NADPH is lower than that of NADH, and that of NADP+ is lower than that of NAD+. Notably, the NADP+-dependent reduction of NAD+ with Fdred is not feasible	Thermotoga maritima	?	-	?

Synonyms

Synonyms	Comment	Organism
NADH-dependent reduced ferredoxin:NADP oxidoreductase	-	Thermotoga maritima
NfnAB	-	Thermotoga maritima

Cofactor

Cofactor	Comment	Organism
FAD	a-FAD and b-FAD, binding of FAD and the iron-sulfur clusters in the NfnAB complex, overview	Thermotoga maritima
Ferredoxin	binding site structure, overview	Thermotoga maritima
additional information	the structure of NfnAB reveals an electron transfer route including the a-FAD, the [2Fe-2S] cluster of NfnA and the b-FAD, and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH*/FAD pair required for ferredoxin reduction. Proposed mechanism of FAD-coupled electron bifurcation by NfnAB, overview	Thermotoga maritima
NADP+	binding site structure, overview	Thermotoga maritima
NADPH	binding site structure, overview	Thermotoga maritima
[2Fe-2S] cluster	iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. Binding of FAD and the iron-sulfur clusters in the NfnAB complex	Thermotoga maritima
[4Fe-4S] cluster	iron-sulfur cluster reconstitution after enzyme purification in 100 mM Tris-HCl, pH 7.5, containing 8 mM DTT, 0.01 mM FAD, 2 mM cysteine, and 1.5 mM FeSO4 at room temperature for 1 h under strictly anaerobic conditions. The proximal [4Fe-4S] cluster is embedded into a rather hydrophilic pocket, and the irons are ligated to three cysteines (Cys51, Cys90, and Cys96) and Glu117. Binding of FAD and the iron-sulfur clusters in the NfnAB complex	Thermotoga maritima

General Information

General Information	Comment	Organism
evolution	NfnA architecturally belongs to the Fnr family, while NfnB is a member of a disulfide oxidoreductase superfamily	Thermotoga maritima