Information on EC 1.13.11.49 - chlorite O2-lyase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
1.13.11.49
-
RECOMMENDED NAME
GeneOntology No.
chlorite O2-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
chloride + O2 = chlorite
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
chlorate reduction
-
-
perchlorate reduction
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-
non-pathway related
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-
SYSTEMATIC NAME
IUBMB Comments
chloride:oxygen oxidoreductase
Reaction occurs in the reverse direction in chlorate- and perchlorate-reducing bacteria. There is no activity when chlorite is replaced by hydrogen peroxide, perchlorate, chlorate or nitrite. The term 'chlorite dismutase' is misleading as the reaction does not involve dismutation/disproportionation. Contains iron and protoheme IX.
CAS REGISTRY NUMBER
COMMENTARY hide
190208-21-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain BC
-
-
Manually annotated by BRENDA team
Alicycliphilus sp.
-
-
-
Manually annotated by BRENDA team
strain GR-1, DSM11199, unclassified
-
-
Manually annotated by BRENDA team
Bacteria GR-1
strain GR-1, DSM11199, unclassified
-
-
Manually annotated by BRENDA team
Dechloromonas sp.
strains JDS5 and JDS6
-
-
Manually annotated by BRENDA team
Dechlorosoma sp.
strain KJ, strain JM, strain HZ, strain PDX. High activity in anaerobically grown bacteria, very low activity in aerobically grown bacteria
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-
Manually annotated by BRENDA team
strain KJ, strain JM, strain HZ, strain PDX. High activity in anaerobically grown bacteria, very low activity in aerobically grown bacteria
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-
Manually annotated by BRENDA team
strai WR341
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-
Manually annotated by BRENDA team
strai WR341
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-
Manually annotated by BRENDA team
gene hemQ
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-
Manually annotated by BRENDA team
gene hemQ
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-
Manually annotated by BRENDA team
from a perchlorate degrading reactor sludge
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
-
the enzyme is the terminal enzyme involved in the perchlorate reduction pathway
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
chloride + O2
chlorite
show the reaction diagram
chlorite
chloride + O2
show the reaction diagram
additional information
?
-
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
chloride + O2
chlorite
show the reaction diagram
chlorite
chloride + O2
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
heme b
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contains 3.7 heme per tetramer
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(NH4)2SO4
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activity increases with increasing salt concentration up to 2 M
CN-
binding kinetics of wild-type and mutant enzymes, overview
Na2SO4
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activity increases with increasing salt concentration up to 2 M
NaCl
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has no significant effect on enzyme activity up to 3.5 g/l
NaClO3
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decreasing activity with increasing salt concentration
NaNO3
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decreasing activity with increasing salt concentration
potassium phosphate
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activity increases with increasing salt concentration up to 1 M salt, decrease beyond 1 M salt
Sodium phosphate
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activity increases with increasing salt concentration up to 1 M salt, decrease beyond 1 M salt
additional information
-
kosmotropic salts such as sulfate and phosphate stabilize protein structures by water-structuring effects of the ions, chaotropic salts such as nitrate and chlorate reduce the activity, no effect of NaCl, NH4Cl, and NaHCO3 (up to 1.5 M) on enzyme activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
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substrate inhibition at higher concentrations over 0.2 mM
azide
chloride
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mixed inhibitor
Chlorite
cyanide
H2O2
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at 70 microM 6% inhibition, at 700 microM 37% inhibtion
hydroxylamine
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complete loss of activity
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.057 - 15.8
Chlorite
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.1 - 200000
Chlorite
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2111 - 600000
Chlorite
2193
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
225
chloride
-
-
0.166
Chlorite
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.982
-
-
2.8
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25C, pH 6.0, 0.2 mM sodium chlorite, culture condition: n-decane/chlorate-grown cell, activity depends on chlorate concentration and amount of cell extract
7.7
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25C, pH 6.0, 0.2 mM sodium chlorite, culture condition: acetate/chlorate-grown cell, activity depends on chlorate concentration and amount of cell extract
443
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Vmax, 100 mM sodium phosphate buffer, pH 6.0, 25C, 0.2 mM chlorite
1500
100 mM K3PO4 (pH 7.0), 25C, oxygen removal by bubbling with high-purity argon, measurement with modified Clarke electrode
4650
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Vmax increase with 1 M phosphate, pH 6.0, 25C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 7.9
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assay at
additional information
-
no pH dependencies in the presence of guaiacol, thioanisole, and monochlorodimedone
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
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activity range
4.5 - 10.5
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activity range, pH-rate profiles and pH-dependent ligand binding constants, active and inactive enzyme forms dependent on the protonation status, steady-state pH-rate profiles, overview
4.7 - 7
Dechlorosoma sp.
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pH 4.8: 45% of maximal activity, pH 7.0: 45% of maximal activity, strain KJ
5 - 8.5
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sodium phosphate buffer, 25C
5.5 - 7
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pH 5.5: about 85% of maximal activity, pH 7.0: about 40% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 25
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assay at
25 - 30
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 45
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100 mM sodium phosphate buffer, pH 7.2
30 - 55
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activity range
30 - 90
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activity range
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.6
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isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
the enzyme does not contain a signal peptide for protein translocation into the periplasm and that the cleaving of a signal peptide at the N terminus would severely disturb the structure of the N-terminal domain
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30420
monomer without heme, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff
31030
monomer with heme, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff
54000
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recombinant mutant W155F, gel filtration
110000
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SDS-PAGE, native enzyme with Superdex 200 column with 50 mM potassium phosphate buffer, pH 7.0 and 100 mM NaCl
115000
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gel filtration
116000
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gel filtration
140000
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gel filtration
155000
pentamer, native mass spectrometry, native mass spectrometry, enzyme buffer-exchanged to 50 mM ammonium acetate, pH 6.8, with centrifugal filter units with 30 kDa cutoff
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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conformational and thermal stability of recombinant dimeric Cld from Nitrobacter winogradskyi, overview
hexamer
homodimer
homopentamer
homotetramer
monomer
pentamer
tetramer
additional information
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the Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi is significantly smaller than all previously known chlorite dismutases
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the enzyme in the natural host contains a covalent modification, probably an intrachain cross-link involving a residue in the 52-55 region and a residue in the 215-223 region. A tyrosine-histidine cross-link appears possible, and can account for EPR differences between the native and recombinant enzymes as well as the spectrophotometric titration of the native enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop method
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thiocyanate inhibited enzyme with incorporated heme, crystals grown from well solution containing 100 mM Mes buffer (pH 5.5), 25% polyethylene glycol monomethyl ether 2000, 0.3 M KSCN, 5% glycerol, 160-260 mM (NH4)2SO4, before data collection crystals are soaked in a solution of the mother liquor including 16% glycerol, crystal is flash-frozen and kept at -173C (100 K) for multiple anomalous dispersion (MAD) with synchrotron radiation
recombinant protein, buffer-exchanged into 20 mM HEPES, pH 7.0, hanging-drop vapor diffusion, 16% PEG 8000, 0.2 M calcium acetate, 90 mM MES, pH 6.5, 293 K, chrystals appear after 2 h, co-crystals with substrate analogues sodium nitrite and sodium cyanide under same conditions
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purified recombinant His-tagged enzyme, sitting-drop vapor diffusion technique and a nanodropdispensing robot, from 1.0 M Na2HPO4-NaH2PO4, pH 8.2, at 22C, 1 week, X-ray diffraction structure determination and analysis at 2.10 A resolution
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purified recombinant His-tagged wild-type and mutant enzymes with bound cyanide, 22C, sitting drop vapour diffusion method, X-ray diffraction structure determination and analysis at 1.85-2.70 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 10
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the pentameric enzyme is very stable between pH 3 and 10 with Tm=92C at pH 7.0
724448
4 - 12
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the activity decreases dramatically below pH 5 and above pH 10, loss of activity is irreversible and accompanied by dramatic and rapid changes in the heme UV/vis chromophore, overview
712251
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
purified recombinant enzyme, inactivation
70
-
purified recombinant enzyme, 36% activity remaining
90
-
purified recombinant enzyme, inactivation
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is capable of approximately 17000 turnovers per heme before undergoing irreversible inactivation due to oxidative damage to the heme
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is capable of approximately 17000 turnovers per heme before undergoing irreversible inactivation due to oxidative damage to the heme
-
724334
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 4 weeks, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell pellet suspended in 0.05 M Tris-HCl buffer, pH 9.5 and 0.05 EDTA for 30 min at room temperature, centrifuged, supernatant ultracentrifuged, red supernatant containing periplasmic fraction separated from pellet, diluted with 10 mM Tris-HCl buffer, pH 7.2, purification with KTAfplc, diluted supernatant loaded onto hydroxyapatite column with 10 mM Tris-HCl, pH 7.2, enzyme elutes at the start of a linear gradient of 10 mM Tris-Hcl, pH 7.2 to 450 mM potassium phosphate, pH 7.2, storage at -20C
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cell-free extracts are prepared from strain AW-1 cells grown in anaerobic medium with n-decane as sole carbon and energy source and chlorate as electron acceptor, centrifugation, storage of extracts under N2 gas at 4C
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centrifuged cell extract, cells grown in AW-1-sulfate medium with benzene and chlorate or benzene and oxygen
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Co2+ chelating resin column chromatography
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HiLoad 16/60 Superdex 200 prep-grade column, equilibrated with 20 mM Tris-HCl (pH 7.5) and 135 mM NaCl to determin molecular Stokes radius
recombinant enzyme from Escherichia coli by anion exchange chromatography and gel filtration
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recombinant His-tagged enzyme from Escherichia coli strain Tuner (DE3) by affinity chromatography
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recombinant His-tagged enzyme from Escherichia coli strain Tuner (DE3) by nickel affinity chromatography and gel filtration
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recombinant mature Cld from Tuner(DE3) Escherichia coli cells by anion exchange chromatography and gel filtration
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recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
recombinant Strep-tagged enzyme from Escherichia coli strain Tuner (DE3) by affinity chromatography, the Strep-II-tag is fully cleaved off using TEV-protease, followed by gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of His-tagged enzyme in Escherichia coli strain Tuner (DE3)
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enzyme gene chemically synthesized without signal sequence, cloned into vector pET28a with histidine tag and thrombin cleavage site (removed before crystallization), and overexpressed
expression in Escherichia coli
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expression in Escherichia coli BL21
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expression in Escherichia coli results in an active enzyme with similar spectral properties as the native enzyme
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expression in Escherichia coli, EPR differences between the native and recombinant enzymes as well as the spectrophotometric titration of the native enzyme
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expression of C-terminally His-tagged enzyme in Escherichia coli strain Tuner (DE3)
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gene hemQ, overexpression in Escherichia coli, complementation of the hemQ-defective Staphylococcus aureus mutant
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phylogenetic analysis, expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
recombinant expression of the enzyme without the N-terminal signal, but with N-terminal TEV-cleavable Strep-II tag in Escherichia coli strain Tuner (DE3)
-
the gene encoding mature Cld, secretion peptide removed and replaced with an N-terminal Met residue, is expressed in untagged form from the pET41a vector in Tuner(DE3) Escherichia coli cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
W155F
-
site-directed mutagenesis, mutation of a conserved residue on the proximal side of the heme causes a loss of the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the wild-type protein. Conversion to an inactive, heme-free form is accelerated by dilution
W156F
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site-directed mutagenesis, mutation of a conserved residue on the proximal side of the heme causes a loss of the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the wild-type protein. Conversion to an inactive, heme-free form is accelerated by dilution
W227F
-
site-directed mutagenesis, mutation of a conserved residue on the proximal side of the heme, but W227F retains many properties of the wild-type protein, the mutant reacts with peracetic acid at pH 6.0-8.0, overview
R173A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, structure determination and comparison to the wild-type enzyme
R173K
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, structure determination and comparison to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
environmental protection
molecular biology
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use of the heme enzyme for the rapid, in situ generation of O2 at concentrations far exceeding 2 mM in coupled enzyme reaction systems to study O consumption or O2 involving reaction steps. Catalytic concentrations of chlorite O2-lyase can be used to initiate the reaction of an O2-utilizing (metallo)enzyme by rapid mixing with the highly soluble, non-volatile ClO2, rather than with the sparingly soluble, gaseous O2, e.g. activation of the beta2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis by mixing its MnII/FeII complex with ClO2- in the presence of chlorite O2-lyase, overview