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Information on EC 1.1.2.3 - L-lactate dehydrogenase (cytochrome) and Organism(s) Saccharomyces cerevisiae and UniProt Accession P00175
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1.1.2.3 L-lactate dehydrogenase (cytochrome)IUBMB Comments
Identical with cytochrome b2; a flavohemoprotein (FMN).
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Word Map
- 1.1.2.3
- flavin
- heme
- phagocyte
-
baker
- hansenula
-
intermembrane
- anomala
- semiquinone
- ferricyanide
-
flavodehydrogenase
-
lederer
- gp91phox
- presequence
-
p40phox
-
heme-binding
-
carbanion
-
mathews
-
flavin-binding
-
chapman
- bromopyruvate
-
flavosemiquinone
- analysis
- medicine
- environmental protection
- synthesis
- diagnostics
The taxonomic range for the selected organisms is: Saccharomyces cerevisiae
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
flavocytochrome b2, cytochrome b2, flavocytochrome b, fc b2, l-lactate cytochrome c oxidoreductase, l-lcr, l-lactate:cytochrome c oxidoreductase, l-lactate-cytochrome c oxidoreductase, l-lactate ferricytochrome c oxidoreductase, l-lactate:cytochrome c-oxidoreductase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
cytochrome b2
-
-
-
-
flavocytochrome b2
L(+)-lactate:cytochrome c oxidoreductase
-
-
-
-
L-lactate cytochrome c oxidoreductase
L-lactate cytochrome c reductase
-
-
-
-
L-lactate dehydrogenase [Cytochrome]
-
-
-
-
L-lactate ferricytochrome C oxidoreductase
L-LCR
-
-
-
-
lactate dehydrogenase (cytochrome)
-
-
-
-
lactic acid dehydrogenase
-
-
-
-
lactic cytochrome c reductase
-
-
-
-
flavocytochrome b2
-
-
-
-
flavocytochrome b2
-
-
L-lactate cytochrome c oxidoreductase
-
-
-
-
L-lactate ferricytochrome C oxidoreductase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
hydride transfer mechanism
(S)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
the hydride transfer mechanism involves the transfer of the lactate hydroxyl proton to H373 while the substrate aH atom is transferred as a hydride to the flavin mononucleotide (FMN) prosthetic group anchored in the active site
(S)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
hydride transfer mechanism
-
(S)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
catalytic cycle, overview
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(S)-lactate:ferricytochrome-c 2-oxidoreductase
Identical with cytochrome b2; a flavohemoprotein (FMN).
CAS REGISTRY NUMBER
COMMENTARY
9078-32-4
-
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
(S)-lactate + 2 flavocytochrome b2
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + cytochrome c
pyruvate + oxidized cytochrome c
-
-
-
?
L-lactate + ferricyanide
pyruvate + ferrocyanide + H+
-
-
-
?
L-lactate + O2
? + superoxide anion
the FDH domain reacts slowly with oxygen with formation of superoxide anion when separated from its natural electron acceptor, whether isolated or included in the holoenzyme
-
-
?
L-lactate + potassium ferricyanide
pyruvate + potassium ferrocyanide + H+
-
-
-
?
(S)-2-hydroxybutyrate + ferricytochrome c
2-oxobutyrate + ferrocytochrome c
-
-
-
-
?
(S)-2-hydroxyhexanoate + ferricytochrome c
2-oxohexanoate + ferrocytochrome c
-
-
-
-
?
(S)-2-hydroxyoctanoate + ferricytochrome c
2-oxooctanoate + ferrocytochrome c
-
-
-
-
?
(S)-2-hydroxyvalerate + ferricytochrome c
2-oxovalerate + ferrocytochrome c
-
-
-
-
?
(S)-lactate + cytochrome c
pyruvate + oxidized cytochrome c
-
-
-
-
?
(S)-phenyllactate + ferricytochrome c
phenylpyruvate + ferrocytochrome c
-
-
-
-
?
glycolate + ferricytochrome c
glyoxylate + ferrocytochrome c
-
very poor substrate
-
-
?
L-lactate + ferricyanide
pyruvate + ferrocyanide + H+
-
-
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
-
?
phenyllactate + ferricytochrome c
phenylpyruvate + ferrocytochrome c
-
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
the first step of the catalytic cycle consists of the oxidation of L-lactate by the FMN prosthetic group. FMN is later reoxidized by transferring its electrons one by one to the ferric heme. The final electron acceptor is cytochrome c
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
flavocytochrome b2, i.e. L-lactate cytochrome c oxidoreductase, catalyzes L-lactate oxidation at the expense of cytochrome c in the mitochondrial intermembrane space in yeast and enables the latter to grow on lactate as the sole carbon source
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
molecular dynamics studies on active-site models of flavocytochrome b2 in complex with the substrate for analysis of the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, overview. In the calculated enzyme-substrate model complex, the l-lactate alpha-OH hydrogen is hydrogen bonded to the activesite base H373 Ne, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by a-OH proton abstraction
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
r
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
r
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
D-isomer not oxidized
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
r
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
r
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
kcat/KM is 24000 fold lower with the recombinantly expressed flavocytochrome b2 flavin-binding domain compared to wild-type enzyme
-
-
?
(S)-mandelate + ferricytochrome c
hydroxy(phenyl)acetate + ferrocytochrome c
-
-
-
-
?
(S)-mandelate + ferricytochrome c
hydroxy(phenyl)acetate + ferrocytochrome c
-
traces of activity with wild type enzyme, significant activity with A198G/L230G double mutant
-
-
?
additional information
?
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
during the catalytic cycle, electrons are transferred one by one from the reduced flavin to heme b2 in the same subunit
-
-
?
additional information
?
-
the role of the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction, of an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, which modulates the reactivity of the key catalytic histidine, and of the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
ferricyanide used as electron acceptor
-
-
?
additional information
?
-
-
2,6-dichloroindophenol, ferricyanide, methylene blue, 1,2-naphthoquinone and cytochrome c can serve as electron acceptors
-
-
?
additional information
?
-
-
2,6-dichlorophenolindophenol used as electron acceptor, 10times more sensitive than ferricyanide
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
additional information
?
-
-
electron acceptors other than ferricytochrome c used
-
-
?
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
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
the first step of the catalytic cycle consists of the oxidation of L-lactate by the FMN prosthetic group. FMN is later reoxidized by transferring its electrons one by one to the ferric heme. The final electron acceptor is cytochrome c
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
-
-
-
-
?
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
flavocytochrome b2, i.e. L-lactate cytochrome c oxidoreductase, catalyzes L-lactate oxidation at the expense of cytochrome c in the mitochondrial intermembrane space in yeast and enables the latter to grow on lactate as the sole carbon source
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
r
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
-
can feed electrons to respiratory chain at the level of cytochrome c
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
the flavoenzyme possesses flavocytochrome b2 subunits, that each consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase, FDH, domain
cytochrome
-
each subunit of the tetramer is composed of two domains, one binding a heme and the other an FMN prosthetic group. The cytochrome domain consists of residues 1 to 99
-
cytochrome c
the flavoenzyme possesses flavocytochrome b2 subunits, that each consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase, FDH, domain
FMN
each of the four subunits contains a flavin mononucleotide prosthetic group
heme
the flavoenzyme possesses flavocytochrome b2 subunits, that each consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase, FDH, domain
heme
each enzyme subunit contains a cytochrome b5-like heme domain
FMN
-
each subunit of the tetramer is composed of two domains, one binding a heme and the other an FMN prosthetic group. The flavin binding domain contains a parallel beta8alpha8 barrel structure and is composed of residues 100 to 486. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent. It forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error
FMN
-
flavohemoprotein, in the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons
heme
-
flavohemoprotein, in the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. The heme domains are mobile in solution
additional information
ferricyanide used as electron acceptor
-
additional information
electron acceptors other than ferricytochrome c used
-
additional information
FMN and heme groups are localized in two different domains
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
electron acceptors other than ferricytochrome c used
-
additional information
-
2,6-dichloroindophenol, ferricyanide, methylene blue, 1,2-naphthoquinone and cytochrome c can serve as electron acceptors
-
additional information
-
2,6-dichlorophenolindophenol used as electron acceptor, 10-fold more sensitive than ferricyanide
-
additional information
-
binding studies and models of the complex between cytochrome c and Fcb2, E63 is involved in the interaction with cyt. c, overview. Cytochrome c mutants E63K/D72K and E63K/D72K show reduced activity, but neither the K296M nor the Y97F mutation show significantly alteration of the enzyme kinetics. Effect of mutations on heme and cytochrome binding, overview
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Fe2+
-
bound to enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
L-lactate
-
60% inhibition at 10 mM, no inhibition with cleaved form of enzyme
pyruvate
-
competitive at low concentrations, non-competitve at high concentrations
additional information
-
enzyme is inhibited by high substrate concentrations
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.02
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P44A
0.032
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67Q
0.051
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant L65A
0.061
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64R
0.065
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
0.069
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64Q
0.085
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67L
0.092
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant N69K
0.097
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant D72A
0.105
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant V70M
0.109
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant K73A
0.113
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
0.119
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant E63K
0.131
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, wild-type enzyme
0.19
(S)-lactate
-
pH 7.5, 25°C, Tris-HCl buffer, mutant Y143F, with heme
0.4
(S)-lactate
-
pH 7.0, 5°C, phosphate buffer, mutant Y143F, with heme
0.53
(S)-lactate
-
pH 7.5, 25°C, Tris-HCl buffer, wild-type enzyme, with heme
0.54
(S)-lactate
-
pH 7.0, 5°C, phosphate buffer, wild-type enzyme, with heme
0.84
(S)-lactate
-
pH 7.5, 25°C, Tris-HCl buffer, wild-type enzyme, with FMN
0.89
(S)-lactate
-
pH 7.0, 5°C, phosphate buffer, wild-type enzyme, with FMN
2.5 - 5
(S)-lactate
-
pH 7.0, 5°C, phosphate buffer, mutant Y143F, with FMN
2.81
(S)-lactate
-
pH 7.5, 25°C, Tris-HCl buffer, mutant Y143F, with FMN
0.0015
ferricytochrome c
-
pH 7.5, 25°C, Tris-HCl buffer, mutant Y143F
0.01
ferricytochrome c
-
pH 7.5, 25°C, Tris-HCl buffer, wild-type enzyme
0.023
ferricytochrome c
-
pH 7.5, 25°C, recombinantly expressed flavocytochrome b2 flavin-binding domain
0.045
ferricytochrome c
-
pH 7.0, 5°C, phosphate buffer, wild-type enzyme
0.121
ferricytochrome c
-
pH 7.0, 30°C, phosphate buffer, mutant Y143F
0.131
ferricytochrome c
-
pH 7.0, 30°C, phosphate buffer, wild-type enzyme
0.131
ferricytochrome c
-
pH 7.0, 5°C, phosphate buffer, mutant Y143F
0.0001
L-lactate
-
Y254L mutant enzyme with 2,6-dichloroindophenol as electron acceptor
0.037
L-lactate
-
wild type enzyme with 2,6-dichloroindophenol as electron acceptor
0.04
L-lactate
-
Y143F mutant enzyme with 2,6-dichloroindophenol as electron acceptor
0.13
L-lactate
-
H373Q mutant enzyme with 2,6-dichloroindophenol as electron acceptor
0.23
L-lactate
-
Y143F mutant with cytochrome c as electron acceptor
0.24
L-lactate
-
native enzyme with cytochrome c as electron acceptor
0.49
L-lactate
-
native enzyme mutant with ferricyanide as electron acceptor
0.66
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7, 30°C
0.66
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7.5, 25°C
0.86
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 10 mM Tris/HCl buffer, 0.1 M NaCl, pH 7.5, 25°C
0.89
L-lactate
-
stopped-flow kinetic parameters for flavin reduction by L-lactate using holo-enzyme, reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, in the absence of ferrocyanide
0.9
L-lactate
-
influence of anions (200 mM phsophate) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
0.94
L-lactate
-
influence of anions (400 mM KBr) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
1.03
L-lactate
-
stopped-flow kinetic parameters for flavin reduction by L-lactate using FDH domain, reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, in the absence of ferrocyanide
1.18
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C
1.5
L-lactate
-
influence of anions (400 mM KCl) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
2.3
L-lactate
-
influence of anions (400 mM KCl) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
2.7
L-lactate
-
influence of anions (400 mM potassium acetate) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
2.8
L-lactate
-
influence of anions (300 mM phsophate) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
4
L-lactate
-
influence of anions (400 mM KBr) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
4.6
L-lactate
-
influence of anions (400 mM potassium acetate) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
5.8
L-lactate
-
influence of anions (400 mM KBr) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
6.5
L-lactate
-
influence of anions (400 mM KCl) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
7
L-lactate
-
mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
8.7
L-lactate
-
influence of anions (200 mM phsophate) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
9.2
L-lactate
-
influence of anions (400 mM potassium acetate) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
additional information
additional information
steady-state kinetics
-
additional information
additional information
stopped-flow, pre-steady-state and steady-state kinetics measuring electron transfer in artificial systems, redox potentials, overview
-
additional information
additional information
-
steady-state kinetic parameters and 2H kinetic isotope effects for the isolated flavin domain and intact, wild-type flavocytoochrome b2
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
270
(S)-lactate
wild-type enzyme with R289 distal, pH 7.0, 30°C
6
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67Q
39
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
62
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
63
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant L65A
87
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P44A
108
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67L
139
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant E63K
143
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64R
155
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, wild-type enzyme
164
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant D72A
165
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant K73A
168
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64Q
171
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant V70M
184
cytochrome c
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant N69K
185
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67Q
190
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant L65A
196
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
207
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64Q
208
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant A67L
209
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant F39A
211
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P44A
212
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant P64R
214
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant D72A
214
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, wild-type enzyme
218
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant E63K
218
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant N69K
223
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant K73A
225
FAD
pH 7.0, 30°C, flavin turnover in pre-steady-state flavin reduction, mutant V70M
0.011
ferricytochrome c
-
pH 7.5, 25°C, Tris-HCl buffer, mutant Y143F
0.02
ferricytochrome c
-
pH 7.5, 25°C, recombinantly expressed flavocytochrome b2 flavin-binding domain
0.103
ferricytochrome c
-
pH 7.5, 25°C, Tris-HCl buffer, wild-type enzyme
61
ferricytochrome c
-
pH 7.0, 5°C, phosphate buffer, wild-type enzyme
155
ferricytochrome c
-
pH 7.0, 30°C, phosphate buffer, wild-type enzyme
7.8
L-lactate
-
influence of anions (400 mM KBr) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
8.6
L-lactate
-
mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
8.8
L-lactate
-
influence of anions (400 mM potassium acetate) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
9.2
L-lactate
-
influence of anions (200 mM phsophate) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
9.2
L-lactate
-
influence of anions (400 mM KCl) on mutant R289K steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 2 mM ferricyanide and variable L-lactate concentration
41
L-lactate
-
A198G/L230G mutant enzyme with ferricyanide as electron acceptor
45
L-lactate
-
influence of anions (400 mM KBr) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
60
L-lactate
-
influence of anions (400 mM KCl) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
61
L-lactate
-
influence of anions (400 mM KBr) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
71
L-lactate
-
influence of anions (200 mM phsophate) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
75
L-lactate
-
influence of anions (400 mM KCl) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
86
L-lactate
-
influence of anions (400 mM potassium acetate) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
101
L-lactate
-
influence of anions (300 mM phsophate) on the FDH domain steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, constant 10 mM ferricyanide and variable L-lactate concentration
103
L-lactate
-
native enzyme with cytochrome c as electron acceptor
113
L-lactate
-
influence of anions (400 mM potassium acetate) on the wild-type steady-state kinetic. Reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, 1.5 mM ferricyanide and variable L-lactate concentration
117
L-lactate
-
holo-enzyme, reaction conditions: 1 or 2 mM ferricyanide, 100 mM phosphate buffer, 1mM EDTA, pH 7, 5°C
133
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C
144
L-lactate
-
stopped-flow kinetic parameters for flavin reduction by L-lactate using holo-enzyme, reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, in the absence of ferrocyanide
149
L-lactate
-
stopped-flow kinetic parameters for flavin reduction by L-lactate using FDH domain, reaction conditions: 100 mM phosphate buffer, 1 mM EDTA, pH 7, 5°C, in the absence of ferrocyanide
214
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 10 mM Tris/HCl buffer, 0.1 M NaCl, pH 7.5, 25°C
240
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7.5, 25°C
259
L-lactate
-
FDH domain, reaction conditions: 13 mM ferricyanide, 100 mM phosphate buffer, 1 mM EDTA, pH 7, 30°C
400
L-lactate
-
native enzyme and Y143F mutant with ferricyanide as electron acceptor
0.02
L-Mandelate
-
wild type enzyme with ferricyanide as electron acceptor
8.5
L-Mandelate
-
A198G/L230G mutant enzyme with ferricyanide as electron acceptor
additional information
additional information
-
systematic determination of substrates with different chain length
-
additional information
additional information
-
systematic determination of activity of all mutants with L-mandelate and L-lactate as substrate
-
additional information
additional information
-
systematic determination of turnover numbers for wild type enzyme and deletion mutants with different electron acceptors, significantly reduced activity for deletion mutants with cytochrome c as electron acceptor
-
additional information
additional information
-
second-order rate constant for cytochrome c reduction in the pre-steady-state determined by stopped-flow spectrophotometry
-
additional information
additional information
-
steady-state kinetic parameters and 2H kinetic isotope effects for the isolated flavin domain and intact, wild-type flavocytoochrome b2
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
170
acetate
-
in pre-steady-state kinetic studies of the FDH domain reduction and reoxidation, acetate acts as a competitive inhibitor of L-lactate
450
chloride
-
in pre-steady-state kinetic studies of the FDH domain reduction and reoxidation, chloride acts as a competitive inhibitor of L-lactate
0.0073
zinc-substituted cytochrome c
-
pH 7.5, 25°C, wild-type enzyme
-
6
pyruvate
-
using the FDH domain at varied ferrocyanide concentrations and a fixed L-lactate concentration pyruvate behaves as a mixed-type inhibitor toward ferrocyanide
6
pyruvate
-
using the FDH domain at varied L-lactate concentrations and a fixed ferrocyanide concentration pyruvate behaves as a mixed-type inhibitor toward L-lactate
9.7
pyruvate
-
in pre-steady-state kinetic studies of the FDH domain reduction and reoxidation, pyruvate acts as a competitive inhibitor of L-lactate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
the pyruvate produced as a result of L-lactate oxidation is utilised by the Krebs cycle, but flavocytochrome b2 also forms part of a short respiratory electron transport chain which results in one ATP molecule being produced for every L-lactate consumed
physiological function
flavocytochrome b2, i.e. L-lactate cytochrome c oxidoreductase, catalyzes L-lactate oxidation at the expense of cytochrome c and enables the latter to grow on lactate as the sole carbon source
physiological function
-
glycolate oxidase3, a glycolate oxidase homolog of yeast L-lactate cytochrome c oxidoreductase, supports L-lactate oxidation in roots of Arabidopsis thaliana
additional information
molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics (QM/MM) scheme to study the mechanism of L-lactate oxidation by flavocytochrome b2. Simulation results highlight the influence of the environment on the catalytic mechanism by describing a step-wise process in the wild-type enzyme with R289 in a distal position and a concerted mechanism for the other systems. Structure analysis of pyruvate in the Fcb2 active site pocket with R289 in the distal conformation. Residue Y254 plays a role in the catalytic process by stabilizing the product of the first proton transfer from substrate to H373, while residue D282 is expected to stabilize the imidazolium ion in transition and product states by electrostatic interactions and hydrogen bonding with the Hdelta of H373. Active site structures of wild-type and mutant enzymes, detailed overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
57500
220000
47000
-
x * 47000, recombinantly expressed flavocytochrome b2 flavin-binding domain, SDS-PAGE
58600
-
4 * 58600, calculated bases on amino acid sequence and heme extinction coefficient
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotetramer
tetramer
?
-
x * 47000, recombinantly expressed flavocytochrome b2 flavin-binding domain, SDS-PAGE
homotetramer
tetramer
homotetramer
enzyme-substrate structure analysis during thr reaction cycle, overview
homotetramer
-
each subunit of the soluble tetrameric enzyme consists of an N-terminal b5-like heme-binding domain and a C terminal flavodehydrogenase. The first 99 residues are folded around the heme, the next about 390 constitute the FMN-binding domain, and the last residues up to 511 make contacts with the other three subunits. Thus, the flavodehydrogenase domains constitute the core of the molecule, with a fourfold symmetry, while the heme domains lie at the periphery
tetramer
-
4 * 58600, calculated bases on amino acid sequence and heme extinction coefficient
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of mutant H723Q bound with pyruvate is determined at 2.8 A
crystallization of flavin binding domain and intact enzyme, hanging drop vapor diffusion method
hanging drop vapor diffusion method, using 20% polyethylene glycol 4000, 0.1 M MES, pH 6.5, and 0.2 M MgCl2, at 18°C
mutant L230A in complex with phenylglyoxalate, mutant A198G/L230A in complex with sulfite, mutant A198G/L230A in complex with pyruvate
crystal structure of flavocytochrome b2 solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering
-
crystallization of Y143F mutant, vapor diffusion technique in presence of PEG 4000
-
Fcb2 free and in complex with sulfite, X-ray diffraction structure analysis at 2.3-2.6 A resolution
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A67L
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
A67Q
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
D282N
site-directed mutagenesis, while the wild-type mutant has residue R289 in a distal or a proximal conformation, the mutant shows R289 only in a distal conformation
D72A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
E63K
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
F39A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
H373Q
K73A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
L230A
L230A/A198G
the double mutant enzyme has a 6fold greater catalytic efficiency with L-mandelate than with L-lactate
L65A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
N69K
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P44A
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P64Q
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
P64R
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
V70M
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
Y254F
the mutant enzyme has a Vmax value some 28fold lower than that of the wild type enzyme and a slightly raised L-lactate Km value
Y254L
site-directed mutagenesis, while the wild-type mutant has residue R289 in a distal or a proximal conformation, the mutant shows R289 only in a distal conformation
Y74F
site-directed mutagenesis, comparison of the mutant kinetics in electron transfer from flavin to heme to the wild-type kinetics
A198G/L230A
A67L
-
reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
A67Q
-
reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
E63K
-
reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
E91K
-
mutation has no effect on the rate of cytochrome c reduction, no significantly different behavior with regard to inhibition by ferrocytochrome c
L230A
N69K
-
reaction proceeds slower than in wild type, no inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
P64Q
-
less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
P64R
-
less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochrome b2
R289K
-
kcat (1/sec) (substrate: L-lactate): 8.6 (in 200 mM phosphate: 9.2, in 400 mM potassium acetate: 8.8, in 400 mM KCl: 9.2, in 400 mM KBr: 7.8), Km (mM) (substrate: L-lactate): 7.0 (in 200 mM phosphate: 8.7, in 400 mM potassium acetate: 9.2, in 400 mM KCl: 6.5, in 400 mM KBr: 5.8). Mutant is not sensitive for excess lactate concentration. In contrast to the wild-type enzyme high concentrations of acetate, phosphate, chloride and bromide show no influence on the mutant enzyme
V70M
-
less inhibition by monoclonal antibody inhibiting electron transfer via flavocytochromb2
Y143F
Y254F
Y254L
additional information
H373Q
His373 acts as an active site base during the oxidation of lactate to pyruvate. The decrease of 3500fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most affected by the mutation. Primary deuterium and solvent kinetic isotope affects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in wild-type. Structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, shows that the orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. Active site residues Arg289, Asp292, and Leu286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively
H373Q
kcat (1/sec) (substrate:lactate): 0.031 (intact protein), 0.057 (flavin domain only), Km (mM) (substrate: lactate): 0.65 (intact protein), 0.25 (flavin domain only)
H373Q
the mutation results in a 34 orders of magnitude decrease in kcat and a slight increase in L-lactate Km
L230A
the mutant flavocytochrome b2 displays increased selectivity for (S)-2-hydroxyoctanoate over L-lactate by a factor of 40 (kcat/Km)
A198G/L230A
-
double mutant enzyme shows significant activity towards L-mandelate
additional information
the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow. This is not unexpected in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core. Reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c
additional information
-
the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow. This is not unexpected in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core. Reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c
additional information
introduction of a number of heme domain side chain substitutions in and around the interface to probe their effect on flavin to heme and cytochrome b2 electron transfer, overview
additional information
active site structures of wild-type and mutant enzymes, detailed overview
additional information
-
three different deletion mutants with deletion of 3, 6 and 9 amino acids from hinge region
additional information
-
for detection of the enzyme activity, recombinant enzyme is immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiratio, overview
additional information
-
enzyme-deficient mutant cells can be complementated by expression of GOX3, a glycolate oxidase from Arabdiospsis thaliana, which is capable to synthesize L-lactate from pyruvate, overview
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, in 0.1 M phosphate buffer, 1 mM EDTA, pH 7, 10 mM DL-lactate is added to the Fcb2 preparations to keep the enzyme in the reduced state
-70°C, purified enzymes are stored in 100 mM potassium phosphate, 1 mM EDTA, and 20 mM D,L-lactate at pH 7.5
-70°C in 0.1 M phosphate buffer, 1 mM EDTA, pH 7, 10 mM DL-lactate is added to the Fcb2 preparations to keep the enzyme in the reduced state
-
4°C, 70% saturated ammonium sulfate, under nitrogen, 2 months, 20% loss of activity of purified enzyme
-
4°C, precipitate from 70% saturated ammonium sulfate, under nitrogen, stable for several weeks
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purification in presence and absence of PMSF results intact or cleaved enzyme
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type Fcb2 and its recombinant FDH domain (FMN-binding domain) are expressed in Escherichia coli
recombinant wild-type Fcb2 and its recombinant flavin domain are expressed in Escherichia coli
expressed in Saccharomyces cerevisiae YN1 strain (flavocytochrome b2 deletion mutant of strain XS560-1)
-
expression of flavocytochrome b2 flavin-binding domain in Escherichia coli
-
overexpression in Hansenula polymorpha leading to enhanced L-lactate-dependent respiration compared to the wild-type cells
-
recombinant wild-type Fcb2 and its recombinant FMN-binding domain (FDH domain) are expressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
flavocytochrome b2 production is induced by the presence of oxygen and L-lactate
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JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
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3
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301
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Saccharomyces cerevisiae
-
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Biochimie
77
621-630
1995
Saccharomyces cerevisiae
Tegoni, M.; Begotti, S.; Cambillau, C.
X-ray structure of two complexes of the Y143F flavocytochrome b2 mutant crystallized in the presence of lactate or phenyl lactate
Biochemistry
34
9840-9850
1995
Saccharomyces cerevisiae
Sharp, R.E.; Chapman, S.K.; Reid, G.A.
Deletions in the interdomain hinge region of flavocytochrome b2: Effects on intraprotein electron transfer
Biochemistry
35
891-899
1996
Saccharomyces cerevisiae
Daff, S.; Ingledew, W.J.; Reid, G.A.; Chapman, S.K.
New insights into the catalytic cycle of flavocytochrome b2
Biochemistry
35
6345-6350
1996
Saccharomyces cerevisiae
Sinclair, R.; Reid, G.A.; Chapman, S.K.
Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of L-mandelate dehydrogenase activity
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333
117-120
1998
Saccharomyces cerevisiae
Mowat, C.G.; Beaudoin, I.; Durley, R.C.; Barton, J.D.; Pike, A.D.; Chen, Z.W.; Reid, G.A.; Chapman, S.K.; Mathews, F.S.; Lederer, F.
Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase)
Biochemistry
39
3266-3275
2000
Saccharomyces cerevisiae
Gondry, M.; Duboist, J.; Terrier, M.; Lederer, F.
The catalytic role of tyrosine 254 in flavocytochrome b2 (L-lactate dehydrogenase from baker's yeast). Comparison between the Y254F and Y254L mutant proteins
Eur. J. Biochem.
268
4918-4927
2001
Saccharomyces cerevisiae
Cunane, L.M.; Barton, J.D.; Chen, Z.W.; Welsh, F.E.; Chapman, S.K.; Reid, G.A.; Mathews, F.S.
Crystallographic study of the recombinant flavin-binding domain of baker's yeast flavocytochrome b(2): comparison with the intact wild-type enzyme
Biochemistry
41
4264-4272
2002
Saccharomyces cerevisiae (P00175)
Le, K.H.; Mayer, M.; Lederer, F.
Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2
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373
115-123
2003
Saccharomyces cerevisiae
Sobrado, P.; Fitzpatrick, P.F.
Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavages are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism
Biochemistry
42
15208-15214
2003
Saccharomyces cerevisiae
Mowat, C.G.; Wehenkel, A.; Green, A.J.; Walkinshaw, M.D.; Reid, G.A.; Chapman, S.K.
Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation
Biochemistry
43
9519-9526
2004
Saccharomyces cerevisiae (P00175), Saccharomyces cerevisiae
Smutok, O.V.; Os'mak, G.S.; Gaida, G.Z.; Gonchar, M.V.
Screening of yeasts producing stable L-lactate cytochrome c oxidoreductase and study of the regulation of enzyme synthesis
Microbiology
75
20-24
2006
Saccharomyces cerevisiae, Ogataea angusta, Saccharomyces cerevisiae IZR-106, Ogataea angusta 356, Saccharomyces cerevisiae IZR-42, Ogataea angusta K-105
-
Boubacar, A.K.; Pethe, S.; Mahy, J.P.; Lederer, F.
Flavocytochrome b2: reactivity of its flavin with molecular oxygen
Biochemistry
46
13080-13088
2007
Saccharomyces cerevisiae (P00175), Saccharomyces cerevisiae
Cenas, N.; Le, K.H.; Terrier, M.; Lederer, F.
Potentiometric and further kinetic characterization of the flavin-binding domain of Saccharomyces cerevisiae flavocytochrome b2. Inhibition by anions binding in the active site
Biochemistry
46
4661-4670
2007
Saccharomyces cerevisiae
Tsai, C.L.; Gokulan, K.; Sobrado, P.; Sacchettini, J.C.; Fitzpatrick, P.F.
Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base
Biochemistry
46
7844-7851
2007
Saccharomyces cerevisiae (P00175)
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Flavin-containing heme enzymes
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493
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2010
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309
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Saccharomyces cerevisiae
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Interaction of cytochrome c with flavocytochrome b2
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35
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Saccharomyces cerevisiae
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48
10803-10809
2009
Saccharomyces cerevisiae (P00175)
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Saccharomyces cerevisiae
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Saccharomyces cerevisiae (P00175)
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110
269-272
2010
Saccharomyces cerevisiae, Saccharomyces cerevisiae D273-10B/A1
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Structural evidence for the functional importance of the heme domain mobility in flavocytochrome b2
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Saccharomyces cerevisiae (P00175), Saccharomyces cerevisiae
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40
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Saccharomyces cerevisiae
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18
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Saccharomyces cerevisiae (P00175)
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Arabidopsis thaliana, Saccharomyces cerevisiae
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