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Information on EC 1.12.99.6 - hydrogenase (acceptor) and Organism(s) Escherichia coli and UniProt Accession P69739
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1.12.99.6 hydrogenase (acceptor)IUBMB Comments
Uses molecular hydrogen for the reduction of a variety of substances. Contains iron-sulfur clusters. The enzyme from some sources contains nickel.
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Word Map
- 1.12.99.6
- hydrogenases
-
h-cluster
- nitrogenase
- reinhardtii
- chlamydomonas
-
diiron
- ferredoxins
-
dithiolate
- desulfovibrio
- hydf
-
hupls
-
subcluster
-
maturase
-
heterocystous
- nostoc
-
organometallic
- eutropha
- acetobutylicum
- pasteurianum
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n2-fixing
-
o2-tolerant
-
diazotrophic
-
photoproduction
-
electrocatalytic
-
electrocatalysts
-
photobiological
- fhl
-
mixed-valence
- gallinae
-
hydrogen-producing
-
biohydrogen
-
overpotential
-
nickel-containing
- cubane
-
diphosphine
-
sacrificial
-
heterolytic
-
oxygen-tolerant
-
hydrogen-oxidizing
-
h2-producing
- desulfuricans
- roseopersicina
-
syntrophic
-
h2-oxidizing
-
photocatalytic
- f0f1-atpase
- h2ases
-
thiocapsa
- industry
- brewing
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
[fefe]-hydrogenase, uptake hydrogenase, hydrogenase 3, hyda1, hyd-1, fe-hydrogenase, hyd-2, hydrogenase 1, nife hydrogenase, hydrogenase 2, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Ech hydrogenase
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-
-
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F420-reducing [NiFe] hydrogenase
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-
-
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factor420 hydrogenase
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-
-
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ferredoxin hydrogenase
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-
-
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H2 producing hydrogenase [ambiguous]
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-
-
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HycE
Hyd-1
Hyd-2
hydrogen dehydrogenase
-
-
-
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hydrogen-lyase
-
-
-
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hydrogen-lyase [ambiguous]
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-
-
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hydrogen:ferredoxin oxidoreductase
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-
-
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hydrogen:methylviologen oxidoreductase
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-
-
-
hydrogenase (ferredoxin)
-
-
-
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hydrogenase 2
hydrogenase 3
hydrogenase-1
hydrogenlyase [ambiguous]
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-
-
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methyl viologen-reducing hydrogenase
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-
-
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methylviologen hydrogenase
-
-
-
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nickel-iron hydrogenase
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-
-
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uptake hydrogenase [ambiguous]
-
-
-
-
hydrogenase 3
-
hydrogenase 3 is the large subunit of a [Ni-Fe]-type hydrogenase
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
-
-
-
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reduction
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-
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PATHWAY SOURCE
PATHWAYS
BRENDA
-
-
SYSTEMATIC NAME
IUBMB Comments
hydrogen:acceptor oxidoreductase
Uses molecular hydrogen for the reduction of a variety of substances. Contains iron-sulfur clusters. The enzyme from some sources contains nickel.
CAS REGISTRY NUMBER
COMMENTARY
9027-05-8
-
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
H2 + 2,3,5-triphenyltetrazolium chloride
reduced 2,3,5-triphenyltetrazolium chloride
-
-
-
-
?
H2 + acceptor
reduced acceptor
the periplasmic-facing membrane-bound complex functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient
-
-
?
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
-
-
-
-
?
additional information
?
-
-
the enzyme exhibits both proton-reducing and H2-oxidizing activities
-
-
?
H2 + acceptor
H+ + reduced acceptor
-
benzyl viologen, methylene blue and methylviologen can act as electron acceptors
-
-
?
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
H2 + acceptor
reduced acceptor
the periplasmic-facing membrane-bound complex functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Fe-S center
the enzyme contains three FeS clusters: a distal [4Fe-4S]2+/1+ center, a medial [3Fe-4S]1+/0 center and a proximal [4Fe-3S]5+/4+ center
[3Fe-4S]-center
the enzyme contains three FeS clusters: a distal [4Fe-4S]2+/1+ center, a medial [3Fe-4S]1+/0 center and a proximal [4Fe-3S]5+/4+ center. The main reason for O2 tolerance is the presence of a special [4Fe-3S] cluster in the proximal position relative to the active site, which discharges a second electron when O2 attacks
[4Fe-3S]-center
the enzyme contains three FeS clusters: a distal [4Fe-4S]2+/1+ center, a medial [3Fe-4S]1+/0 center and a proximal [4Fe-3S]5+/4+ center
-
[4Fe-4S]-center
the enzyme contains three FeS clusters: a distal [4Fe-4S]2+/1+ center, a medial [3Fe-4S]1+/0 center and a proximal [4Fe-3S]5+/4+ center
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ni2+
Ni2+
-
accessory protein HypB is necessary for Ni(II) incorporation into the hydrogenase protein. HypB has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
N-bromosuccinimide
-
complete inactivation at 10 mM, 20% inactivation at 0.1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
HypB
-
accessory protein HypB is necessary for the production of active hydrogenase
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.008
-
hydrogen uptake specific activity of the hydrogenase 3 lacking mutant enzyme HD705/pBS(Kan), using methyl viologen as a substrate
0.035
-
hydrogen uptake specific activity of the wild type enzyme, using methyl viologen as a substrate
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
growth of a strain that lacks a functional fructose phosphotransferase system in the presence of fructose results in an approximately 10fold increase in isoform Hyd1 levels in comparison with growth under the same conditions with glucose. This increase in the amount of Hyd1 is not due to regulation at the transcriptional level. Reintroduction of a functional fructose phosphotransferase system restores growth on D-fructose and reduces Hyd1 levels to those observed after growth on D-glucose. Reducing the rate of glucose uptake by introducing a mutation in the gene encoding the cAMP receptor protein, or consumption through glycolysis, by introducing a mutation in phosphoglucose isomerase, increased Hyd1 levels during growth on glucose. Thus the ability to oxidize hydrogen by Hyd1 shows a strong correlation with the rate of carbon flow through glycolysis and provides a direct link between hydrogen, carbon and energy metabolism
physiological function
additional information
HybO availability is an important limiting factor for native Hyd-2 synthesis
physiological function
growth of a strain that lacks a functional fructose phosphotransferase system in the presence of fructose results in an approximately 10fold increase in isoform Hyd1 levels in comparison with growth under the same conditions with glucose. This increase in the amount of Hyd1 is not due to regulation at the transcriptional level. Reintroduction of a functional fructose phosphotransferase system restores growth on D-fructose and reduces Hyd1 levels to those observed after growth on D-glucose. Reducing the rate of glucose uptake by introducing a mutation in the gene encoding the cAMP receptor protein, or consumption through glycolysis, by introducing a mutation in phosphoglucose isomerase, increased Hyd1 levels during growth on glucose. Thus the ability to oxidize hydrogen by Hyd1 shows a strong correlation with the rate of carbon flow through glycolysis and provides a direct link between hydrogen, carbon and energy metabolism
physiological function
-
during lactose fermentation by Escherichia coli mainly Hyd-4 and partially Hyd-3 affect cell growth and H2 production at different pHs. Hyd-3 and Hyd-4 are responsible for H2 production at pH 7.5, 6.5 and 5.5
physiological function
-
endogenous hydrogenases Hyd-1 and Hyd-2 induce respective 26.5 % and 36.7 % increases of succinate yield, indicating that both Hyd-1 and Hyd-2 contribute to the increased pool of reductive equivalents
physiological function
the periplasmic-facing membrane-bound complex functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient. HybO availability is an important limiting factor for native Hyd-2 synthesis
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
64000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein (HybB)
tetramer
the membrane-extrinsic part of hydrogenase-1 is a dimer of heterodimers (alphabeta)2
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
to 3.3 A resolution, in a 2:1 complex with its physiological partner, cytochrome b. From the short distance between distal [Fe4S4] clusters, a rapid transfer of H2-derived electrons between hydrogenase heterodimers is predicted. Thus, under low O2 levels, a functional active site in one heterodimer can reductively reactivate its O2-exposed counterpart in the other
sitting drop vapor diffusion technique, the crystal structure of recombinant enzyme is solved to a maximum resolution of 1.5 A (reduced) or 2.2 A (as-isolated)
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D202V/K492
-
variant epHycE70, has 11fold higher hydrogen production and 7fold higher hydrogen yield from formate compared to wild-type
D210N/I271F/K545R
-
variant epHycE23-2, has 8fold higher hydrogen production and 4fold higher hydrogen yield from formate compared to wild-type
F297L/L327Q/E382K/L415M/A504T/D542N
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variant epHycE17, has 7fold higher hydrogen production and 4fold higher hydrogen yield from formate compared to wild-type
I333F/K554d
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variant epHycE39, has 7fold higher hydrogen production and 3fold higher hydrogen yield from formate compared to wild-type
Q32R/V112L/G245C/F409L
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variant epHycE21, has 15fold higher hydrogen production and 6fold higher hydrogen yield from formate compared to wild-type
S2P/E4G/M314V/T366S/V394D/S397C
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variant epHycE67, has 13fold higher hydrogen production and 5fold higher hydrogen yield from formate compared to wild-type
S2T/Y50F/I171T/A291V/T366S/V433L/M444I/L523Q
T366
Y464
-
variant shufHycE1-9, has 23fold higher hydrogen production and 9fold higher hydrogen yield from formate compared to wild-type
additional information
S2T/Y50F/I171T/A291V/T366S/V433L/M444I/L523Q
-
shows a 17fold higher hydrogen-producing activity and 8fold higher hydrogen yield from formate than wild type HycE
S2T/Y50F/I171T/A291V/T366S/V433L/M444I/L523Q
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variant epHycE95, has 17fold higher hydrogen-producing activity and 8fold higher hydrogen yield from formate compared to wild-type
T366
-
variant satHycE12T366, has 30fold higher hydrogen production compared to wild-type
T366
-
variant satHycE19T366, has 27fold higher hydrogen production compared to wild-type
additional information
-
saturation mutagenesis at T366 of HycE leads to increased hydrogen production via a truncation at this position, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30fold
additional information
-
the uptake activity in mutant HD705/pBS(Kan), which is defective in hydrogenase 3, is 4.4fold lower than that in wild-type enzyme, the hydrogen uptake activity in the hydrogenase 1 and 2 double mutant (hyaB hybC) is reduced 2.7fold by addition of the hycE mutation (hyaB hybC hycE)
additional information
changing a tyrosine or threonine, located on the protein surface within 10 A of the distal [4Fe-4S] and medial [3Fe-4S] clusters, to cysteine, allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H2 production. Assemblies of Y227C (or T225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H2 production at a TiO2 electrode. A colloidal system for visible-light photo-H2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Eachhydrogenase produces 40 molecules of H2 per second and sustains 20% activity in air
additional information
-
changing a tyrosine or threonine, located on the protein surface within 10 A of the distal [4Fe-4S] and medial [3Fe-4S] clusters, to cysteine, allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H2 production. Assemblies of Y227C (or T225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H2 production at a TiO2 electrode. A colloidal system for visible-light photo-H2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Eachhydrogenase produces 40 molecules of H2 per second and sustains 20% activity in air
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 10
-
activity of hydrogenase I is progressively lost between pH 7 and pH 10, hydrogenase II is not affected
439630
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
90% loss of activity after 8 days in the presence of oxygen, increasing ionic strength causes 10-40% reversible inhibition
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE Sepharose column chromatography and Q Sepharose HiLoad column chromatography
-
HisTrap Ni affinity column chromatography, and Superdex 200 gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
an overexpression system is described that facilitates the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex
variants expressed from epPCR and DNA shuffling plasmid pBS(Kan)HycE in Escherichia coli BW25113 hyaB hybC hycE DELTAkan
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
-
endogenous hydrogenases Hyd-1 and Hyd-2 can be utilized to engineer H2 in a microbial cell factory manner
synthesis
-
hydrogenases Hyd-1 and Hyd-2 are engineered to enable the cells to efficiently utilize hydrogen gas as a source of reducing equivalents, which increased the yield of reduced fermentation products, e.g. succinate or lactate. By upregulating the expression of the hydrogenase, the spectrum of fermentation products shifts toward the reductive end compared to that of the wild type
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ballantine, S.P.; Boxer, D.H.
Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12
J. Bacteriol.
163
454-459
1985
Escherichia coli
Sawers, R.G.; Boxer, D.H.
Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12
Eur. J. Biochem.
156
265-275
1986
Escherichia coli
Maeda, T.; Sanchez-Torres, V.; Wood, T.K.
Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities
Appl. Microbiol. Biotechnol.
76
1035-1042
2007
Escherichia coli
Maeda, T.; Sanchez-Torres, V.; Wood, T.K.
Protein engineering of hydrogenase 3 to enhance hydrogen production
Appl. Microbiol. Biotechnol.
79
77-86
2008
Escherichia coli, Escherichia coli K-12 BW25113
Forzi, L.; Hellwig, P.; Thauer, R.K.; Sawers, R.G.
The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins
FEBS Lett.
581
3317-3321
2007
Escherichia coli
Dias, A.V.; Mulvihill, C.M.; Leach, M.R.; Pickering, I.J.; George, G.N.; Zamble, D.B.
Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB
Biochemistry
47
11981-11991
2008
Escherichia coli
Pinske, C.; McDowall, J.S.; Sargent, F.; Sawers, R.G.
Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12
Microbiology
158
856-868
2012
Escherichia coli (P0ACD8), Escherichia coli (P69739), Escherichia coli
Volbeda, A.; Darnault, C.; Parkin, A.; Sargent, F.; Armstrong, F.A.; Fontecilla-Camps, J.C.
Crystal structure of the O(2)-tolerant membrane-bound hydrogenase 1 from Escherichia coli in complex with its cognate cytochrome b
Structure
21
184-190
2013
Escherichia coli (P69739), Escherichia coli
Flanagan, L.A.; Wright, J.J.; Roessler, M.M.; Moir, J.W.; Parkin, A.
Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance
Chem. Commun. (Camb.)
52
9133-9136
2016
Escherichia coli
Pinske, C.; Jaroschinsky, M.; Linek, S.; Kelly, C.L.; Sargent, F.; Sawers, R.G.
Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli
J. Bacteriol.
197
296-306
2015
Escherichia coli
Yang, C.; Li, Z.; Zhao, D.; Chen, J.; Zhu, X.; Zhang, X.; Bi, C.
Engineering an efficient H2 utilizing Escherichia coli platform by modulation of endogenous hydrogenases
Biochem. Eng. J.
166
107851
2021
Escherichia coli, Escherichia coli ATCC 8739
-
Beaton, S.E.; Evans, R.M.; Finney, A.J.; Lamont, C.M.; Armstrong, F.A.; Sargent, F.; Carr, S.B.
The structure of hydrogenase-2 from Escherichia coli implications for H2-driven proton pumping
Biochem. J.
475
1353-1370
2018
Escherichia coli (P69741), Escherichia coli K12 (P69741)
Mirzoyan, S.; Trchounian, A.; Trchounian, K.
Role of hydrogenases 3 and 4 in Escherichia coli growth and H2 producing hydrogenase activity during anaerobic utilization of lactose
Int. J. Hydrogen Energy
43
18151-18159
2018
Escherichia coli, Escherichia coli BW25113, Escherichia coli MC 4100
-
Zhang, L.; Morello, G.; Carr, S.B.; Armstrong, F.A.
Aerobic photocatalytic H2 production by a [NiFe] hydrogenase engineered to place a silver nanocluster in the electron relay
J. Am. Chem. Soc.
142
12699-12707
2020
Escherichia coli (P0ACD8), Escherichia coli, Escherichia coli K12 (P0ACD8)
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