Information on EC 1.16.5.1 - ascorbate ferrireductase (transmembrane)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.16.5.1
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RECOMMENDED NAME
GeneOntology No.
ascorbate ferrireductase (transmembrane)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ascorbate[side 1] + Fe(III)[side 2] = monodehydroascorbate[side 1] + Fe(II)[side 2]
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
Fe(III):ascorbate oxidorectuctase (electron-translocating)
A diheme cytochrome that transfers electrons across a single membrane, such as the outer membrane of the enterocyte, or the tonoplast membrane of the plant cell vacuole. Acts on hexacyanoferrate(III) and other ferric chelates.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ascorbate[side 1] + Fe(CN)3[side 2]
monodehydroascorbate[side 1] + ?
show the reaction diagram
ascorbate[side 1] + Fe(III)-citrate[side 2]
?
show the reaction diagram
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-
-
-
?
ascorbate[side 1] + Fe(III)-EDTA[side 2]
?
show the reaction diagram
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-
-
-
?
ascorbate[side 1] + Fe(III)[side 2]
monodehydroascorbate[side 1] + Fe(II)[side 2]
show the reaction diagram
ascorbate[side 1] + Fe3+-EDTA[side 2]
monodehydroascorbate[side 1] + ?
show the reaction diagram
ascorbate[side 1] + ferricyanide[side 2]
?
show the reaction diagram
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-
-
-
?
ascorbate[side 1] + nitroblue tetrazolium[side 2]
dehydroascorbate + ?
show the reaction diagram
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-
-
-
?
bathocuprionedisulfonate[side 1] + Cu(II)-nitrilotriacetic acid[side 2]
Cu(I)-bathocuproinedisulfonate
show the reaction diagram
-
-
-
-
?
cupric-histidine[side 1] + ?
?
show the reaction diagram
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-
-
-
?
ferric citrate[side 1] + ?
?
show the reaction diagram
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-
-
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?
ferrozine[side 1] + Fe(III)-nitrilotriacetic acid[side 2]
Fe(II)-ferrozine
show the reaction diagram
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-
-
-
?
ferrozine[side 1] + Fe3+-EDTA[side 2]
?
show the reaction diagram
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-
-
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ascorbate[side 1] + Fe(III)[side 2]
monodehydroascorbate[side 1] + Fe(II)[side 2]
show the reaction diagram
cupric-histidine[side 1] + ?
?
show the reaction diagram
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-
-
-
?
ferric citrate[side 1] + ?
?
show the reaction diagram
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-
-
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
cytochrome b561
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heme b
additional information
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Diethylpyrocarbonate
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the electron accepting ability of bovine cytochrome b561 from ascorbate is selectively inhibited by the treatment with diethylpyrocarbonate
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
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cytb561 ferric reductase activity is greatly enhanced by the addition of ascorbate
dehydroascorbate
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preloading cells with dehydroascorbate greatly increases both ferric and cupric reductase activities of Dcytb
L-galactono-gamma-lactone
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0152 - 0.0231
Cu(II)-nitrilotriacetic acid[side 2]
0.074 - 0.0921
Fe(III)-nitrilotriacetic acid[side 2]
additional information
additional information
stopped-flow kinetic analysis at pH 5.0-7.0
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
oxidation rate by monodehydroascorbate radical
6
reduction rate by ascorbate
additional information
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
the parasitic trematode Schistosoma japonicum contains a CYB561 protein localized to the schistosome tegument
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
of duodenal mucosa
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26900
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calculated from amino acid sequence
27000
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LCytb, SDS-PAGE
27800
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CGCytb, SDS-PAGE
30000
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SDS-PAGE
31500
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DCytb, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 25000, about, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetylation
the N-terminus of isozyme CBCytB is anchored to the membrane by acetylation of the amino-terminal Met residue
no glycoprotein
although the deduced amino acid sequence of Zmb561 contains two potential N-glycosylation sites (109NES111 and 203NFT205), periodic acid-Schiff staining of the purified wild-type-Zmb561 in SDS-PAGE gels do not show any indication of glycosyl groups
additional information
the N-terminal Met residue is removed posttranslationally. No partial proteolytic digestion at the C-terminal part of wild-type Zmb561
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states is reported. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native chromaffin granule CYB561 from adrenal gland from the chromaffin granule membrane by Triton X-100 solubilzation and separation of the solubilized nCGCytb by preparative electrophoresis. Recombinant C-terminaly His6-tagged isozyme CGCytb from Spodoptera frugiperda Sf9 cells, Pichia pastoris strain GS115, or Escherichia coli by nickel affinity chromatography to homogeneity
native enzyme from microsomal membranes by anion exchange and concanavalin A affinity chromatography, recombinant His6-tagged enzyme from Pichia pastoris by anion exchange and nickel affinity chromatography and gel filtration, elution with a solution containing 1.0% w/v n-octyl beta-glucoside
purified by affinity chromatography and gel filtration
recombinant C-terminally His6-tagged isozyme DCytb from Spodoptera frugiperda Sf9 cells, and untagged, apoform or fully functional isozyme DCytb from Escherichia coli, to homogeneity
recombinant C-terminaly His6-tagged enzyme from yeast YPH499 cells by nickel affinity chromatography, recombinant C-terminally His6-tagged isozyme DCytb from Escherichia coli
recombinant untagged four CYB561 isoforms from yeast YPH499 cells partially, two recombinant C-terminally His10- or Strep-II-tagged CYB561 paralogues from Escherichia coli and Pichia pastoris by affinity chromatography
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and recombinant expression of untagged four CYB561 isoforms in yeast YPH499 cells, recombinant expression of two C-terminally His10- or Strep-II-tagged CYB561 paralogues in either Escherichia coli or in Pichia pastoris. Functional expression of the enzyme in Saccharomyces cerevisiae strain S288C DELTAfre1DELTAfre2 deficient in ferric reductase activity
Dcytb-EGFP is expressed in tetracycline-off Madin-Darby canine kidney cells
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expressed as a His-tagged fusion protein in Escherichia coli
expressed in a Saccharomyces cerevisiae strain S288C DELTAfre1DELTAfre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity
expressed in Escherichia coli
expressed in Saccharomyces cerevisiae, strain YPH499
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expressed in the Saccharomyces cerevisiae mutant strain S288C DELTAfre1DELTAfre2 that lacks plasma membrane ferrireductase activity
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expressed in Xenopus laevis oocytes
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functional expression of the enzyme in Saccharomyces cerevisiae strain S288C DELTAfre1DELTAfre2 deficient in ferric reductase activity
functional expression of the enzyme in Saccharomyces cerevisiae strain S288C DELTAfre1DELTAfre2 which is deficient in ferric reductase activity
recombinant expression of C-terminally His6-tagged isozyme CGCytb in Spodoptera frugiperda Sf9 cells as well as in Pichia pastoris strain GS115, and recombinant expression of C-terminally His6-tagged isozyme CGCytb in Escherichia coli
recombinant expression of C-terminally His6-tagged isozyme DCytb in Spodoptera frugiperda Sf9 cells, untagged, apoform or fully functional isozyme DCytb in Escherichia coli
recombinant expression of C-terminaly His6-tagged enzyme in yeast YPH499 cells, recombinant expression of C-terminally His6-tagged isozyme DCytb in Escherichia coli
recombinant expression of His6-tagged enzyme in Pichia pastoris under control of a methanol-inducible promoter AOX1
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of Dcytb-EGFP is knocked down by addition of 20 ng/ml doxycycline
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iron-deficient patients have significantly higher ferric reductase activity and duodenal and plasma ascorbate concentrations than do control subjects
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F105W/H106E
mutations on the noncytoplasmic side only still allows the oxidized Cyt b561 to be reduced by ascorbate
H117A
site-directed mutagenesis, the mutation leads to reduced reduction of ascorbate by the mutant TCytb
H156A
site-directed mutagenesis, the mutation leads to reduced reduction of ascorbate by the mutant TCytb
H50A
site-directed mutagenesis, the mutation leads to reduced reduction of ascorbate by the mutant TCytb
H83A
site-directed mutagenesis, the mutation leads to reduced reduction of ascorbate by the mutant TCytb
H83A/H156A
site-directed mutagenesis
H83L/H156L
site-directed mutagenesis
K81A/R150A
mutations on the cytoplasmic side only still allows the oxidized Cyt b561 to be reduced by ascorbate
K81A/R150A/F105W/H106E
Y115W
mutations on the noncytoplasmic side only still allows the oxidized Cyt b561 to be reduced by ascorbate
Y140W
mutations on the cytoplasmic side only still allows the oxidized Cyt b561 to be reduced by ascorbate
E79A
site-directed mutagenesis, the mutation in bovine rCGCytb causes significant (but no extreme) alteration in at least one of the two (sometimes three) midpoint ascorbate concentrations characterizing the redox transition of hemes-b, and the mutation does not block the reduction of either heme-b center
N78K
site-directed mutagenesis, the mutation in bovine rCGCytb does not influence the physicochemical properties of protein as compared to the wild-type
T84A
site-directed mutagenesis, the mutation in bovine rCGCytb causes significant (but no extreme) alteration in at least one of the two (sometimes three) midpoint ascorbate concentrations characterizing the redox transition of hemes-b, and the mutation does not block the reduction of either heme-b center
H120A
site-directed mutagenesis of DCytb, the mutation results in partial loss of hemes
H159A
site-directed mutagenesis of DCytb, the mutation results in partial loss of hemes
H33A
site-directed mutagenesis, mutation in human DCytb does not influence the physicochemical properties of protein as compared to the wild-type
H50A
site-directed mutagenesis of DCytb, the mutation results in complete loss of hemes
H50A/H120A
site-directed mutagenesis of DCytb, the mutant contains one heme-b per double His-mutant rDCytb
H86A
site-directed mutagenesis of DCytb, the mutation results in complete loss of hemes
H86A/H159A
site-directed mutagenesis of DCytb, the mutant contains one heme-b per double His-mutant rDCytb
D38A
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the mutant shows increased Fe(CN)3 reductase activity compared to the wild type enzyme
E117A
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the mutant shows decreased Fe(CN)3 reductase activity compared to the wild type enzyme
E196A
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the mutant shows decreased Fe(CN)3 reductase activity compared to the wild type enzyme
F44A
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the mutation reduces the Fe(CN)3 reductase activity by about 45%
H105A
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the mutant shows increased Fe(CN)3 reductase activity compared to the wild type enzyme
H108A
site-directed mutagenesis, the mutation results in a practically unchanged level of protein expression and a considerably lower ascorbate reducibility
H112A
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the mutant shows decreased Fe(CN)3 reductase activity compared to the wild type enzyme
H117A
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the mutation completely abrogates the Fe(CN)3 reductase activity of the enzyme
H120A
site-directed mutagenesis, the mutation results in nearly undetectable levels of rCGCytb
H156A
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the mutation completely abrogates the Fe(CN)3 reductase activity of the enzyme
H159A
site-directed mutagenesis, the mutation results in a practically unchanged level of protein expression and a considerably lower ascorbate reducibility
H47A
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the mutation completely abrogates the Fe(CN)3 reductase activity of the enzyme
H52A
site-directed mutagenesis, the mutation results in nearly undetectable levels of rCGCytb
H86A
site-directed mutagenesis, the mutation results in a practically unchanged level of protein expression and a considerably lower ascorbate reducibility
H86A/H159A
site-directed mutagenesis, the mutation results in a practically unchanged level of protein expression and a considerably lower ascorbate reducibility
M51A
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the mutant shows increased Fe(CN)3 reductase activity compared to the wild type enzyme
N106A
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the mutant shows increased Fe(CN)3 reductase activity compared to the wild type enzyme
P48A
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the mutant shows increased reductase activity compared to the wild type enzyme
Q131A
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the activity of the mutant is reduced significantly to 45% of that of the wild type
R149A
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the mutation results in a 75% loss in activity compared to the wild type enzyme
R67A
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the mutation results in an almost complete loss of the Fe(CN)3 reductase activity
R72A
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
R72E
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type; site-directed mutagenesis, the mutation of TCytb does not affect the final reduction level of rTCytb by ascorbate but results in a complete loss of the pH-dependent initial time-lag upon electron acceptance from ascorbate
R72K
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
R72T
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
R72Y
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
S115A
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the ferrireductase activity in the mutant is reduced to 50%
W119A
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the ferrireductase activity in the mutant is reduced to 17%; the mutant shows decreased Fe(CN)3 reductase activity compared to the wild type enzyme
Y190A
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the mutant shows increased Fe(CN)3 reductase activity compared to the wild type enzyme
Y66A
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the mutation results in an almost complete loss of the Fe(CN)3 reductase activity
additional information
replacing any of the 4 highly conserved His residues, coordinating the two b-type hemes, by Ala in mouse rLCytb completely abolishes the transmembrane ferric reductase activity of rLCytb. Midpoint ascorbate concentration for the reduction of low-potential heme-b centers is hardly influenced by the R74X replacements but that for the high-potential heme-b centers show a significant trend