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Information on EC 1.3.7.7 - ferredoxin:protochlorophyllide reductase (ATP-dependent) and Organism(s) Rhodobacter capsulatus and UniProt Accession P26164
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1.3.7.7 ferredoxin:protochlorophyllide reductase (ATP-dependent)IUBMB Comments
Occurs in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms. The enzyme catalyses trans-reduction of the D-ring of protochlorophyllide; the product has the (7S,8S)-configuration. Unlike EC 1.3.1.33 (protochlorophyllide reductase), light is not required. The enzyme contains a [4Fe-4S] cluster, and structurally resembles the Fe protein/MoFe protein complex of nitrogenase (EC 1.18.6.1), which catalyses an ATP-driven reduction.
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Word Map
- 1.3.7.7
-
light-dependent
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rhodobacter
- capsulatus
- angiosperms
- nitrogenase
- bacteriochlorophyll
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gymnosperm
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nitrogenase-like
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phototrophs
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dark-grown
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nb-protein
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anoxygenic
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marchantia
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etioplasts
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prolamellar
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light-grown
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dinitrogen
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plectonema
- boryanum
- liverwort
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photosynthesis-related
The taxonomic range for the selected organisms is: Rhodobacter capsulatus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
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Synonyms
light-independent protochlorophyllide reductase, dark protochlorophyllide reductase, light-independent pchlide reductase, dark-operative pchlide oxidoreductase, light-independent (dark-operative) pchlide oxidoreductase, dark-operative protochlorophyllide reductase, protochlorophyllide oxidoreductase complex, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dark-operative protochlorophyllide oxidoreductase
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DPOR
light-independent protochlorophyllide reductase
DPOR
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DPOR consists of two components, the L-protein and the NB-protein
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate = protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate = protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
reaction mechanism for trans-specific reduction, overview
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chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate = protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
reaction mechanism and structure-function relationship, overview
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PATHWAY SOURCE
PATHWAYS
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-, -
SYSTEMATIC NAME
IUBMB Comments
ATP-dependent ferredoxin:protochlorophyllide-a 7,8-oxidoreductase
Occurs in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms. The enzyme catalyses trans-reduction of the D-ring of protochlorophyllide; the product has the (7S,8S)-configuration. Unlike EC 1.3.1.33 (protochlorophyllide reductase), light is not required. The enzyme contains a [4Fe-4S] cluster, and structurally resembles the Fe protein/MoFe protein complex of nitrogenase (EC 1.18.6.1), which catalyses an ATP-driven reduction.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
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-
-
?
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
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-
-
-
?
chlorophyllide a + reduced ferredoxin + ATP
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
-
-
-
-
?
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate
-
-
-
-
?
protochlorophyllide a + reduced ferredoxin + 4 ATP + 4 H2O
chlorophyllide a + oxidized ferredoxin + 4 ADP + 4 phosphate
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-
-
?
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
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-
-
-
?
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
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-
-
?
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
-
DPOR catalyzes the stereo-specific reduction of C17-C18 double bond on the D-ring of protochlorophyllide
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-
?
additional information
?
-
The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe-4S]-center, yielding a negatively-charged intermediate. Depending on the rate of Fe-S cluster re-reduction, the reaction either proceeds through double protonation of the single-electron-reduced substrate, or by alternating proton/electron transfer. The Fe-S cluster rereduction should be the rate-limiting stage of the process
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-
?
additional information
?
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-
although chlorophyllide c binds to the substrate-binding pocket in the NB-protein, the C17-C18 double bond on the D-ring of chlorophyllide c is not reduced by the DPOR
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-
?
additional information
?
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DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer)
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-
?
additional information
?
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each catalytic BchN-BchB unit contains one protochlorophyllidee and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific protochlorophyllide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit, unique trans-specific reduction mechanism in which the distorted C17-propionate of protochlorophyllid and an aspartate from BchB serve as proton donors for C18 and C17 of protochlorophyllide, respectively, overview
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-
?
additional information
?
-
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the C175C18 double bond of chlorophyll c is not reduced by DPOR
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
-
-
-
?
chlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
protochlorophyllide + oxidized ferredoxin + 2 ADP + 2 phosphate + 2 H+
-
-
-
-
?
protochlorophyllide + oxidized ferredoxin + ADP + phosphate
chlorophyllide a + reduced ferredoxin + ATP
-
-
-
?
protochlorophyllide a + reduced ferredoxin + 2 ATP + 2 H2O
chlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
[4Fe-4S]-center
reaction potential of the FeS cluster shows pH-dependence
4Fe-4S-center
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NB-protein of DPOR carries two oxygen-tolerant [4Fe4S] clusters
4Fe-4S-center
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the L-protein carries a [4Fe-4S] cluster between the protomers that is very similar to that of the nitrogenase Fe protein. The NB-protein also carries a [4Fe-4S] cluster that mediates electrons from the L-cluster to the protochlorophyllide molecule
Ferredoxin
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each catalytic BchN-BchB unit contains one protochlorophyllidee and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Mg2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Dithionite
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dark protochlorophyllide reductase activity is dependent on the reductant dithionite
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0106
protochlorophyllide
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in 100 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 5 mM dithiothreitol, at 34°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.026
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purified recombinant protochlorophyllide-bound NB-protein, recovered from crystals, pH not specified in the publication, temperature not specified in the publication
0.0262
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purified enzyme, pH not specified in the publication, at 34°C
0.05
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purified recombinant protochlorophyllide-free NB-protein, recovered from crystals, pH not specified in the publication, temperature not specified in the publication
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
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cyanobacteria, algae, bryophytes, pteridophytes and gymnosperms use an additional, light-independent enzyme dubbed dark-operative Pchlide oxidoreductase for chlorophyll biosynthesis, besides a light-dependent enzyme, mechanisms of protochlorophyllide a reduction in photosynthetic organisms, ooverview
metabolism
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the three-subunit enzyme dubbed DPOR operates in the synthesis of Bchls a, b, and g
physiological function
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DPOR performs reduction of the C17-C18 double bond of protochlorophyllide to form chlorophyllide a, the direct precursor of chlorophyll a in a light-independent, dark-operative way of action
additional information
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the organism contains another type of Chl, bacteriochlorophyll (Bchl) a, as compared to Chl a and Chl b of higher plants. Residue Asp36 is not necessary for enzyme complex formation but for enzyme activity. Subunit BchB possesses a unique C-terminal region consisting of approximately 100 amino acid residues (Phe422-Arg525), which is probably important for protochlorophyllide reduction
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
33413
-
2 * 33413, L-protein of DPOR, calculated from amino acid sequence
46038
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2 * 57191 + 2 * 46038, NB-protein of DPOR, calculated from amino acid sequence
48671
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2 * 48671 + 2 * 57191, subunit BchN and subunit BchB, calculated from amino acid sequence
57191
57191
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2 * 48671 + 2 * 57191, subunit BchN and subunit BchB, calculated from amino acid sequence
57191
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2 * 57191 + 2 * 46038, NB-protein of DPOR, calculated from amino acid sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterooctamer
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(alpha2)2(betagamma)4, DPOR is a nitrogenase-like enzyme consisting of two components, L-protein, a BchL dimer, and NB-protein, a BchN-BchB heterotetramer, which are structurally related to nitrogenase Fe protein and MoFe protein, respectively
heterotetramer
homodimer
additional information
heterotetramer
-
2 * 48671 + 2 * 57191, subunit BchN and subunit BchB, calculated from amino acid sequence
heterotetramer
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2 * 57191 + 2 * 46038, NB-protein of DPOR, calculated from amino acid sequence
homodimer
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2 * 33413, L-protein of DPOR, calculated from amino acid sequence
homodimer
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2 * 36046, subunit BchL calculated from amino acid sequence
additional information
-
each catalytic BchN-BchB unit contains one protochlorophyllide and one iron-sulfur NB-cluster coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific protochlorophyllide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit, unique trans-specific reduction mechanism in which the distorted C17-propionate of protochlorophyllide and an aspartate from BchB serve as proton donors for C18 and C17 of protochlorophyllide, respectively, overview
additional information
-
DPOR consists of two components: a reductase component designated L-protein (a BchL dimer) and a catalytic component named NB-protein (a BchN-BchB heterotetramer), structure analysis and comparison to the nitrogenase complex, overview. The NB-cluster is unique because it is coordinated by three Cys residues from BchN (BchN-Cys26, BchN-Cys51, BchN-Cys112) and one Asp residue from BchB (BchB-Asp36)
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
catalytic component NB-protein, both in thePchlide-bound and Pchlide-free states, X-ray diffraction structure determination at 2.3 A and 2.8 A resolution, respectively
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hanging drop vapor diffusion method, the protochlorophyllide-bound form of NB-protein is crystallized using 200 mM sodium/potassium phosphate buffer (pH 5.0) containing 5 mM dithiothreitol and 10% (w/v) ethylene glycol at 4°C, to which 16% (w/v) and 14% (w/v) PEG4K are added in aerobic and anaerobic conditions, respectively, as precipitants. Protochlorophyllide -free and selenomethionine-substituted recombinant NB-proteins are crystallized at 20°C using 20% (w/v) PEG3350 containing 200 mM ammonium chloride and 5mM dithiothreitol. D36C and D36A variants are crystallized at 4°C using 20% (w/v) PEG3350 containing 200 mM sodium chloride, 100 mM MOPS/NaOH (pH 7.0) and 5 mM dithiothreitol as a precipitant
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purified recombinant protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein of DPOR, and purified recombinant selenomethionine-substituted protochlorophyllide-free forms of mutants D36A and D36C, hanging-drop vapour diffusion method., X-ray diffraction structure determination and analysis at 2.3-2.9 A resolution
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C51A
-
the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme)
D36A
D36C
D36S
D36A
-
site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme
D36A
-
the mutant exhibits low activity (13% compared to the wild type enzyme)
D36A
-
site-directed mutagenesis, the mutant subunit B forms a complex with subunit N, indicating that Asp36 is not necessary for complex formation, D36A retains only 13% of wild-type activity
D36C
-
site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme
D36C
-
the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme)
D36C
-
site-directed mutagenesis, the mutant subunit B forms a complex with subunit N, indicating that Asp36 is not necessary for complex formation, catalytically inactive mutant
D36S
-
site-directed mutagenesis, mutation in BchB, mutant NB-cluster structure compered to the wild-type enzyme
D36S
-
the mutation almost abolishes the activity of the enzyme (less than 5% compared to the wild type enzyme)
D36S
-
site-directed mutagenesis, the mutant subunit B forms a complex with subunit N, indicating that Asp36 is not necessary for complex formation, catalytically inactive mutant
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the NB-protein activity persists after the exposure to air for almost 2 h
-
686758
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, NB- and L-protein subunits of the crude extract when maintained anaerobically, more than 6 months, remain stable with no significant loss of activity
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4°C, NB- and L-protein subunits of the purified extract when maintained anaerobically, both activities of the components are rapidly lost during purification procedures such as affinity chromatography with S-protein agarose
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
protochlorophyllide-saturated NB-protein from DPOR is purified by Strep-Tactin Sepharose column chromatography
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recombinant protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein from Rhodobacter capsulatus strain DB176 and Escherichia coli strain JM105 by affinity chromatography and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
protochlorophyllide-bound and protochlorophyllide-free forms of NB-protein are overexpressed in Rhodobacter capsulatus strain DB176 and Escherichia coli strain JM105
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subunits BchL and BchN are expressed in Rhodobacter capsulatus strains YCN1 or YCL3 as S tag fusion proteins, subunit BchB is co-purified with the BchN protein
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Fujita, Y.; Bauer, C.E.
Reconstitution of light-independent protochlorophyllide reductase from purified bchl and BchN-BchB subunits. In vitro confirmation of nitrogenase-like features of a bacteriochlorophyll biosynthesis enzyme
J. Biol. Chem.
275
23583-23588
2000
Rhodobacter capsulatus
Nomata, J.; Swem, L.R.; Bauer, C.E.; Fujita, Y.
Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus
Biochim. Biophys. Acta
1708
229-237
2005
Rhodobacter capsulatus
Nomata, J.; Ogawa, T.; Kitashima, M.; Inoue, K.; Fujita, Y.
NB-protein (BchN-BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe-S clusters
FEBS Lett.
582
1346-1350
2008
Rhodobacter capsulatus
Kondo, T.; Nomata, J.; Fujita, Y.; Itoh, S.
EPR study of 1Asp-3Cys ligated 4Fe-4S iron-sulfur cluster in NB-protein (BchN-BchB)2 of a dark-operative protochlorophyllide reductase complex
FEBS Lett.
585
214-218
2011
Rhodobacter capsulatus
Burke, D.H.; Alberti, M.; Hearst, J.E.
bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants
J. Bacteriol.
175
2414-2422
1993
Rhodobacter capsulatus (D5ANS3), Rhodobacter capsulatus (P26163), Rhodobacter capsulatus (P26164), Rhodobacter capsulatus, Rhodobacter capsulatus SB1003 (D5ANS3)
Muraki, N.; Nomata, J.; Ebata, K.; Mizoguchi, T.; Shiba, T.; Tamiaki, H.; Kurisu, G.; Fujita, Y.
X-ray crystal structure of the light-independent protochlorophyllide reductase
Nature
465
110-114
2010
Rhodobacter capsulatus, Rhodobacter capsulatus DB176
Reinbothe, C.; El Bakkouri, M.; Buhr, F.; Muraki, N.; Nomata, J.; Kurisu, G.; Fujita, Y.; Reinbothe, S.
Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction
Trends Plant Sci.
15
614-624
2010
Cereibacter sphaeroides, Chlorobaculum tepidum, Chloroflexus aurantiacus, Heliobacterium mobile, Prochlorococcus marinus, Rhodobacter capsulatus
Nomata, J.; Terauchi, K.; Fujita, Y.
Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus
Biochem. Biophys. Res. Commun.
470
704-709
2016
Rhodobacter capsulatus (D5ANS3 and P26164 and P26163), Rhodobacter capsulatus ATCC BAA-309 (D5ANS3 and P26164 and P26163)
Silva, P.J.
With or without light comparing the reaction mechanism of dark-operative protochlorophyllide oxidoreductase with the energetic requirements of the light-dependent protochlorophyllide oxidoreductase
PeerJ
2
e551
2014
Rhodobacter capsulatus (P26164), Rhodobacter capsulatus ATCC BAA-309 (P26164)
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