HYPOTHESIS AND THEORY ARTICLE
published: 07 August 2012
doi: 10.3389/fmicb.2012.00273
Bacterial oxygen production in the dark
Katharina F. Ettwig*, Daan R. Speth, Joachim Reimann, Ming L. Wu, Mike S. M. Jetten and Jan T. Keltjens
Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Nijmegen, Netherlands
Edited by:
Boran Kartal, Radboud University,
Netherlands
Reviewed by:
Natalia Ivanova, Lawrence Berkeley
National Laboratory, USA
Carl James Yeoman, Montana State
University, USA
*Correspondence:
Katharina F. Ettwig, Department of
Microbiology, Institute for Water and
Wetland Research, Radboud
University Nijmegen,
Heyendaalseweg 135, 6525 AJ
Nijmegen, Netherlands.
e-mail: k.ettwig@science.ru.nl
Nitric oxide (NO) and nitrous oxide (N2 O) are among nature’s most powerful electron
acceptors. In recent years it became clear that microorganisms can take advantage of
the oxidizing power of these compounds to degrade aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the “NC10” phylum bacterium “Candidatus
Methylomirabilis oxyfera” and the γ-proteobacterial strain HdN1 it has been suggested
that under anoxic conditions with nitrate and/or nitrite, monooxygenases are used for
methane and hexadecane oxidation, respectively. No degradation was observed with
nitrous oxide only. Similarly, “aerobic” pathways for hydrocarbon degradation are employed
by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite (ClO−
2 ).
In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen
formation, substrate activation in the presence of nitrite was directly associated with both
oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an
oxygen species derived from it, in the activation reaction of methane. Although oxygen
generation elegantly explains the utilization of “aerobic” pathways under anoxic conditions, the underlying mechanism is still elusive. In this perspective, we review the current
knowledge about intra-aerobic pathways, their potential presence in other organisms, and
identify candidate enzymes related to quinol-dependent NO reductases (qNORs) that might
be involved in the formation of oxygen.
Keywords: oxygen production, nitric oxide, nitric oxide reductase, chlorate reduction, chlorite dismutase, Cld,
“Candidatus Methylomirabilis oxyfera”, strain HdN1
INTRODUCTION
In dim anoxic waters of stratified lakes where oxygen-respiring
organisms normally cannot survive, a tiny aerobic eukaryote
nevertheless makes a living. This heterotrophic ciliate, Histiobalantium natans, can survive without external oxygen because
it sequesters chloroplasts from ingested euglinoid flagellates
(Phacus suecicus). The chloroplasts, kept active in the ciliate
and surrounded by the mitochondria, photosynthesize and produce oxygen that allows the host to thrive in deep waters of
stratified lakes, where it avoids metazoan predation and competition with other aerobic ciliates (Esteban et al., 2009). This
is just one example of nature’s many twists that allow organisms to take a specific niche: If an essential compound is not
available, make it yourself by inventing a variation on a general
theme.
For a long time, photosynthesis was the only biological process known to produce oxygen. Cyanobacteria, green plants,
and algae use light energy to split water (E0′ = +0.82 V) via
photosystem II. The electrons obtained serve NADPH and ATP
generation for carbon dioxide fixation; oxygen is a mere byproduct of this metabolism. This pathway evolved at least 2.7
billion years ago (Canfield, 2005), and, after the vast pools
of reduced compounds on early earth were exhausted, oxygen
started to accumulate in the atmosphere around 2.45 billion
years ago (Holland, 2006). As a consequence, organisms evolved
numerous mechanisms to cope with and/or exploit its strong
oxidative properties. To prevent oxidative damage by reactive
oxygen species (ROS) like superoxide (O2 − ), hydrogen peroxide
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(H2 O2 ), or the most damaging of all, the hydroxyl radical (OH·),
detoxification systems, which often result in the regeneration of
oxygen (e.g., by catalase or superoxide dismutase) evolved. These
reactions have been studied and reviewed in detail elsewhere (Apel
and Hirt, 2004; Murphy, 2009) and are beyond the scope of
this article.
On the other hand, a large number of extant organisms are
completely dependent on oxygen as the terminal electron acceptor
in aerobic respiration. In addition, oxygen is the substrate in an
enormous variety of monooxygenase and dioxygenase reactions
where one or two oxygen atoms, respectively, are incorporated
into the substrate, either for degradation of compounds like aromatic and aliphatic hydrocarbons or biosynthesis of secondary
metabolites.
De novo oxygen production can be driven by either light or
chemical energy. The second, “dark” way takes advantage of oxidants with a more positive redox potential than the O2 /H2 O
couple. Only a few redox couples are biologically relevant in
this respect: hypochlorite (ClO− )/Cl− ; E0′ = +1.31 V, chlo−
′
′
−
−
rite (ClO−
2 )/ClO (E0 = +1.28 V), ClO2 /Cl (E0 = +1.08 V),
′
nitrous oxide (N2 O)/N2 (E0 = +1.36 V), nitric oxide (NO)/N2 O
(E0′ = +1.18 V), and NO/N2 (E0′ = +1.27 V). Most of these
compounds are intermediates in the respiration of (per)chlorate
and nitrate/nitrite, respectively. In this perspective, we review
what is known and still to be learned about oxygenic pathways from chloro-oxo species and nitrogen oxides, with a focus
on a hypothetical enzymatic mechanism for the hitherto elusive
nitrite-driven oxygen production.
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Ettwig et al.
Bacterial oxygen production in the dark
OXYGEN PRODUCTION IN CHLORATE-REDUCING
BACTERIA
The first group of oxygenic chemotrophs identified were perchlorate and chlorate respiring bacteria (Rikken et al., 1996; van Ginkel
et al., 1996). These organisms reduce perchlorate (ClO4 − ) and/or
chlorate (ClO3 − ) to chlorite (ClO−
2 ). Rather than being further
reduced to hypochlorite (ClO− ), chlorite is converted into chloride (Cl− ) and O2 . Perchlorate occurs naturally, but rarely in the
environment, with significant concentrations only found in the
Chilean salpeter deposits (Beckurts, 1886; Ericksen, 1983). In
past decades, anthropogenic contamination from either the use
of Chile salpeters as fertilizers, or from chemical waste (e.g., solid
rocket fuel spills and explosives) has been a concern and incentive
for research on microbial (per)chlorate reduction (Motzer, 2001;
Xu et al., 2003). An initial surprise was the wide-spread occurrence
of (per)chlorate reduction among microorganisms and in different ecosystems, much broader than could be expected from the
known natural sources and the short timeframe of anthropogenic
contamination (Coates et al., 1999). It now has become clear that
perchlorate is continuously generated in trace amounts in the
atmosphere. Accumulation to measurable amounts, however, only
occurs where deposition is high, but leaching and microbial reduction is low: in an extremely arid climate (Rajagopalan et al., 2006;
Kounaves et al., 2010).
(Per)chlorate respiration in principal only requires two
enzymes. At first, (per)chlorate reductase, a molybdopterincontaining respiratory reductase resembling nitrate reductase,
catalyzes the reduction of perchlorate to chlorate and of chlorate to chlorite. Then, chlorite is converted in a single exergonic
reaction into chloride and oxygen (Eq. 1; van Ginkel et al., 1996).
This is a net disproportionation or dismutase reaction in which
the chlorine atom becomes reduced and oxygen oxidized.
′
−
0
ClO−
= − 100 kJ mol−1 O2 )
2 → Cl + O2 (ΔG
(1)
The reaction is catalyzed at a high rate and with extraordinary
specificity by chlorite dismutases (Cld, EC 1.13.11.49), members of the recently defined CDE superfamily of heme enzymes
(Goblirsch et al., 2011). This homohexameric or homopentameric
heme b enzyme was first purified in the nineties from the βproteobacterium Azospira oryzae (then called strain GR-1, van
Ginkel et al., 1996). The enzyme is now well characterized by
the resolution of the atomic structures from several species (de
Geus et al., 2009; Mehboob et al., 2009b; Goblirsch et al., 2010;
Kostan et al., 2010; Mlynek et al., 2011). Detailed kinetic analysis established that oxygen is not derived from water, but
from chlorite itself (Lee et al., 2008; Streit and DuBois, 2008).
Catalysis proceeds via an oxoferryl species and a ClO− anion,
indicating that, after initial binding to the catalytic heme b, chlorite is first cleaved, after which both oxygen atoms recombine,
yielding chloride and oxygen. Most chlorate-reducing organisms found thus far are facultative aerobes that, in absence of
extracellular oxygen use the chlorite-derived oxygen for aerobic
respiration, analogous to the use of chloroplasts by H. natans in the
introduction.
What else to do with oxygen produced by chorite dismutation? Surprisingly, functional chlorite dismutases have also been
Frontiers in Microbiology | Evolutionary and Genomic Microbiology
found in Bacteria and Archaea that cannot grow with chlorate
as electron acceptor, e.g., in the nitrite-oxidizing genera Nitrospira
(Maixner et al., 2008) and Nitrobacter (Mlynek et al., 2011). In
the latter species, Cld is significantly smaller and present as
a homodimer. The role of Cld in these organisms, however,
is unclear. They may possibly confer insensitivity to chlorite,
coupled with the advantage of producing oxygen for nitrite
oxidation in microoxic niches. In the archaeon Haloferax volcanii, chlorite dismutase is hypothesized to produce oxygen for
a monooxygenase encoded in the same operon, that is involved
in the biosynthesis of an antibiotic (Bab-Dinitz et al., 2006). In
Pseudomonas chloritidismutans, oxygen likely does not only act as
an electron acceptor in respiration, but is also used for alkane activation by a monooxygenase-mediated reaction (Mehboob et al.,
2009a). P. chloritidismutans is capable of respiring carbon substrates like fatty acids or alcohols with oxygen, chlorate, and
nitrate, but growth on alkanes is not observed with nitrate as
electron acceptor. This suggests that oxygen, provided externally
or from chlorate reduction, is required for the initial activation of the alkane, a hydroxylation to the corresponding alcohol
(Heider, 2007).
The β-proteobacterium Dechloromonas aromatica strain RCB
can degrade benzene aerobically and anaerobically with chlorate
and nitrate (Coates et al., 2001). However, signature genes of
anaerobic hydrocarbon activation (Heider, 2007), like the glycylradical enzyme benzyl-succinate synthase cluster, are missing. In
contrast, the genome of strain RCB only encodes genes for the aerobic activation of aromatic compounds, including several monoand dioxygenases (Salinero et al., 2009). Physiological experiments
under nitrate-reducing conditions strongly suggest the involvement of a hydroxyl radical-mediated activation leading to phenol
as primary intermediate (Chakraborty and Coates, 2005). It is
quite unlikely that the very substrate-specific Cld can catalyze O2
production from nitrogen oxide intermediates. This possibility
has been negatively tested for NO with the recombinant Cld of
Nitrospira defluvii (Maixner et al., 2008), which was also found to
be inhibited by NO (179 µM; F. Maixner and K. Ettwig, unpublished results). The open question is: Can the oxidative power for
the attack on benzene come from oxygen, also under denitrifying
conditions (Weelink et al., 2010)?
OXYGEN PRODUCTION FROM NITROGEN OXIDES?
The idea that oxygen may be an intermediate of denitrifying,
anaerobic bacteria emerged when the genome of the anaerobic methane-oxidizing bacterium “Candidatus Methylomirabilis
oxyfera” was assembled from enrichment culture metagenomes.
These freshwater enrichment cultures (Raghoebarsing et al.,
2006; Ettwig et al., 2009) couple complete methane oxidation with
CO2 as the end product to the reduction of nitrite (NO2 − ) to
dinitrogen (N2 ) according to Eq. 2.
+
3 CH4 + 8 NO−
2 + 8 H → 3 CO2 + 4 N2
′
+ 10 H2 O (ΔG0 = −928 kJ mol−1 CH4 )
(2)
Methane has the second highest activation energy (after benzene) of all organic compounds. One of the prime questions
was how it could be enzymatically activated under anaerobic
August 2012 | Volume 3 | Article 273 | 2
Ettwig et al.
A NO –
3
Bacterial oxygen production in the dark
nar
B
NO2–
nir
nir
NO2–
2NO
NO
nor
nos
N2O
N2
N2
nod
r
no
O2
N2O
MO
CO2
alcohol
s
no
alkane
N2
FIGURE 1 | Pathways of canonical denitrification (A) and proposed N2 and
O2 production by NO dismutation (B). nar, nitrate reductase; nir, nitrite
reductase; nor, nitric oxide reductase; nos, nitrous oxide reductase; nod,
nitric oxide dismutase, MO, monooxygenase.
conditions. Generally, two enzymatic activation mechanisms
were already known: Aerobic methane-oxidizing bacteria (MOB)
employ a monooxygenase reaction yielding methanol as the first
intermediate (Hakemian and Rosenzweig, 2007; Trotsenko and
Murrell, 2008). Anaerobic methanotrophic archaea (ANME),
that couple methane oxidation to sulfate reduction (most likely
performed in association with sulfate-reducing bacteria) reverse
the last step of methanogenesis catalyzed by methyl-coenzyme
M reductase (Knittel and Boetius, 2009; Scheller et al., 2010).
Though energetically costly, a third possibility has also been considered – an activation mechanism involving addition of the
methyl group to fumarate, catalyzed by a glycyl radical enzyme
homologous to benzyl- or alkyl-succinate synthase (Thauer and
Shima, 2008).
Whereas no homologues of the two last mentioned signature genes for anaerobic methane and hydrocarbon degradation
could be identified in the genome, surprisingly the entire pathway of aerobic methane oxidation, starting with particulate
methane monooxygenase (pMMO), was present, and prominently transcribed and expressed (Ettwig et al., 2010; Luesken
et al., 2012). This was consistent with several experimental findings: The M. oxyfera enrichment culture was not sensitive to
aerobic handling (Ettwig et al., 2009), but highly sensitive to acetylene (total inhibition at 10 µM; Ettwig et al., 2010), a known
inhibitor of pMMO (Prior and Dalton, 1985). Besides methane,
the M. oxyfera enrichment culture also oxidized other shortchain alkanes (ethane, propane, butane), a well-known activity
of pMMO (Leadbetter and Foster, 1960; Hazeu and de Bruyn,
1980). Finally, using the oxidation of propylene as a proxy for
pMMO activity (Prior and Dalton, 1985), comparable rates were
obtained for oxygen and nitrite as electron acceptors (Ettwig et al.,
2010). Also the analysis of the denitrification pathway caused
surprise. In all microbial species studied so far, denitrification
proceeds in a step-wise fashion, comprising the subsequent reduction of nitrate (NO3 − ) to nitrite (NO2 − ), nitric oxide (NO),
nitrous oxide (N2 O), and eventually dinitrogen gas (N2 ) by dedicated reductases (Figure 1A; Zumft, 1997; Einsle and Kroneck,
2004; Tavares et al., 2006). The last step, nitrous oxide reduction, is not always present, leaving the potent greenhouse gas
N2 O as the end product (Stein, 2011). Thus, a second startling
finding was the apparent lack of an identifiable nitrous oxide
‘Ca M. oxyfera’ Twente DAMO_2434
98 ‘Ca M. oxyfera’ Ooij 2434 consensus
‘Ca M. oxyfera’ Twente DAMO_2437
96
99 ‘Ca M. oxyfera’ Ooij 2437 consensus
69
Gammaproteobacterium HdN1 NorZ2
M. ruestringensis NorZ
96
99
97
1
Gammaproteobacterium HdN1 NorZ1
qNOR
NOR
N. gonorrhoeae NorZ
HCO
Synechocystis sp. NorZ
S. aureus NorZ
53
87
G. stearothermophilus NorZ
‘Ca M. oxyfera’ DAMO_1889
P. aeruginosa NorB
86
P. denitrificans NorB
P. aeruginosa CcoN
100
FIGURE 2 | Maximum likelihood tree (500 bootstrap replicates)
of selected protein sequences from the heme-copper oxidases
(HCO) superfamily, with a focus on quinol-dependent nitric oxide
reductases (qNORs) including the potential NO dismutases
(in red). The tree was calculated with MEGA5 (Tamura et al., 2011) and
is based on an alignment created with ClustalW using the default
settings. The alignment was manually checked for correct alignment
of conserved residues. The sequences Ooij 2434 and Ooij 2437 are
consensus sequences based on the contigs obtained by iterative read
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P. denitrificans CcoN
cNOR
cbb3 oxidase
mapping of the Ooij metagenome (SRR022748.2) to the genome
of M. oxyfera (Dutilh et al., 2009). Accession numbers: M. oxyfera
DAMO_2434 CBE69496, DAMO_2437 CBE69502, and DAMO_1889
CBE68939; γ-proteobacterium HdN1 NorZ1 CBL45628 and NorZ2
YP_003809511; M. ruestringensis G2PJH6; N. gonorrhoeae ZP_04723508;
Synechocystis sp. BAA18795; S. aureus EGL94648; G. stearothermophilus
3AYF_A; P. aeruginosa NorB NP_249215, P. denitrificans NorB BAA32546,
P. aeruginosa CcoN NP_252822; P. denitrificans CcoN
AAC44516.
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Ettwig et al.
Bacterial oxygen production in the dark
FIGURE 3 | Ribbon representation of the overall qNOR structure of
Geobacillus stearothermophilus (3AYG, Matsumoto et al., 2012) seen
from the plane of the cytoplasmic membrane (between dotted lines).
The heme groups are indicated in red, the quinol analog 2-heptyl
hydroxyquinoline N-oxide in magenta. The zinc in the catalytic center is
shown in green, and the calcium (Ca2+ ) assisting in coordination of the
heme groups in pastel green.
reductase in the genome of M. oxyfera, even though it had
been shown that dinitrogen gas was the end product of nitrite
reduction. Despite the presence of three qNOR paralogs (see
below), of which two were highly transcribed and expressed,
nitrous oxide was not produced in significant amounts. Now,
one of two possibilities might explain the paradoxical results: (1)
activation of methane to methanol by NO, yielding N2 as the
second product of the pMMO-catalyzed reaction, (2) the disproportionation of NO into N2 and O2 (Eq. 3), analogously to
the chlorite dismutase reaction and to be catalyzed by a novel
enzyme, NO dismutase (NOD). Again, such disproportionation
is exergonic.
′
2NO → N2 + O2 (ΔG0 = − 173 kJ mol−1 O2 )
(3)
Although the first possibility cannot be discarded, it is very
unlikely. The pMMO of M. oxyfera shows a high sequence identity
to the well-studied enzymes from other organisms with known
Frontiers in Microbiology | Evolutionary and Genomic Microbiology
crystal structures, including amino acids implicated in catalysis
(Balasubramanian and Rosenzweig, 2007; Smith et al., 2011). With
NO as the direct oxidizing agent, at least some modifications
would be expected. Moreover, such role of NO is not in accordance with the experimental reaction stoichiometry (Eq. 2). Next,
the bypass of N2 O as an intermediate, and the formation of
labeled oxygen from 18 O-labeled nitrite could be experimentally
shown (Ettwig et al., 2010). The hypothetical pathway that is consistent with all observations is shown in Figure 1B. From the
overall reaction stoichiometry (Eq. 2) it is inferred that the disproportionation of eight NO molecules would give four oxygen
molecules only three of which are consumed in the activation
of methane. Residual O2 appears to be respired by one of the
terminal oxidases found in the M. oxyfera genome (Wu et al.,
2011). Obviously, the most interesting question now is the identity of the enzyme that catalyzes oxygen and nitrogen formation
from NO.
The intermediary role for oxygen in the activation of recalcitrant compounds during denitrification may not be limited
to M. oxyfera. The facultatively denitrifying γ-proteobacterium
strain HdN1 grows on a wide variety of substrates, including
C6- to C20-alkanes (Ehrenreich et al., 2000; Zedelius et al., 2010).
Growth on hexadecane was observed with oxygen, nitrate, or
nitrite as electron acceptors, but not with N2 O. In contrast, N2 O
did serve as a substrate for growth on the corresponding easier-todegrade C16-alcohol and fatty acid, which do not require oxidative
activation (Zedelius et al., 2010). Like M. oxyfera, the HdN1
genome did not contain recognizable genes for the glycyl-radicalcatalyzed activation of alkanes, such as alkylsuccinate synthase.
Instead, two or possibly three monooxygenases were encoded in
the genome. These findings suggest that the activation of the
alkane substrate in M. oxyfera and HdN1 take place by a similar mechanism involving oxygen, formed from nitrate or nitrite
(Figure 1B).
DIVERGENT NITRIC OXIDE REDUCTASES IN M. OXYFERA
AND OTHER DENITRIFYING MICROORGANISMS
Like oxygen, NO is a strongly oxidizing compound and most
microorganisms that have to deal with it as an intermediate or
in their environment have developed a repertory of enzymes that
convert it into the harmless N2 O as fast as possible (Richardson,
2000; de Vries and Schröder, 2002; Watmough et al., 2009). Collectively, the bacterial nitric oxide reductases (NORs) belong to the
superfamily of heme-copper oxidases (HCOs; Figure 2). Members
of the family share the presence of a heme b (or a) for electron
transfer, and a second heme (b3 , a3 , or o3 ), that together with an
iron (FeB in NOR) or a copper ion (CuB in oxidases) constitute the
catalytic center. Both FeB and CuB are ligated to three conserved
histidines. The electron-transferring heme is coordinated by two
histidines as well, while one more histidine serves as the proximal
ligand to the catalytic heme. This histidine sextet is a signature
for HCOs.
Nitric oxide reductases catalyze the two-electron reduction of
two molecules of NO into N2 O (Eq. 4).
2NO + 2H+ + 2e− → N2 O + H2 O
(4)
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Ettwig et al.
FIGURE 4 | Quinol-binding and catalytic sites in the qNOR structure
of Geobacillus stearothermophilus (3AYG, Matsumoto et al., 2012; left),
and amino acid sequence comparison of these sites in qNORs and
putative NODs (right). Sequence accession numbers and alignment
are as indicated in Figure 2. Numbering above the alignment refers to
the first amino acid and corresponds to the residue numbers of G.
stearothermophilus. Specific changes in otherwise strongly conserved
The different NOR types are distinguished on the basis of
the electron carrier that supplies nitric oxide reduction with
reductant. Best characterized are cNORs which contain an additional cytochrome c subunit for this purpose, and qNORs which
use reduced quinone (quinol) as the electron donor. Of both
enzymes, atomic structures have been resolved recently (Hino
et al., 2010; Matsumoto et al., 2012).
As mentioned above, the M. oxyfera genome contained three
qNOR paralogs (EC 1.7.5.2, DAMO_1889, DAMO_2434, and
DAMO_2437), in stark contrast to the lack of appreciable
N2 O production during nitrite-dependent methane oxidation
(Raghoebarsing et al., 2006; Ettwig et al., 2008, 2009, 2010).
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Bacterial oxygen production in the dark
residues are highlighted. (A) Quinol binding site with a bound quinol analog,
2-heptyl hydroxyquinoline N-oxide (green molecular surface). His328 and
Asp746 form hydrogen bonds with the quinol moiety and the large
hydrophobic residues interact with the hydrophobic tail. (B) View of the
catalytic site from the plane of the heme b3 . The ZnB is indicated in green
and two water molecules in the coordination sphere of the ZnB are indicated
as small red spheres.
DAMO_1889 was expressed in only low amounts, but the
two highly similar DAMO_2434 and DAMO_2437 (84% aa
identity) were among the most abundant gene products,
both at the transcriptional and protein level (Ettwig et al.,
2010). Detailed sequence analysis revealed that DAMO_1889
shared all important features with known qNORs, while
DAMO_2434 and DAMO_2437 displayed important differences,
which will be discussed in detail below. Strikingly, the unusual
characteristics were consistently found in two other protein
sequences available in GenBank, putative qNORs from the
hexadecane-oxidizing γ-proteobacterial strain HdN1 (Zedelius
et al., 2010) and from Muricauda ruestringensis, a Flavobacterium
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Bacterial oxygen production in the dark
that had been isolated with peptone as a carbon source from
a hexadecane-oxidizing, denitrifying enrichment culture (Bruns
et al., 2001). A species of the same genus, M. aquimarina, was
recently shown to degrade hexadecane and polycyclic aromatic
hydrocarbons aerobically (Jiménez et al., 2011). Although the three
organisms are only distantly related, their unusual qNOR-like
genes form one separate cluster within the qNORs (Figure 2).
A similar qNOR, however, is absent from the genome of the
benzene-oxidizing D. aromatica strain RCB.
CHARACTERISTICS OF THE PUTATIVE NO DISMUTASES
The overall atomic structure of qNOR strongly resembles the
one of cNORs and other HCOs (Hino et al., 2010; Matsumoto
et al., 2012). The enzyme is composed of a membrane-spanning
region with 13 trans-membrane helices (TMHs) that enclose the
heme b, heme b3 , and FeB moieties, which are coordinated by the
conserved histidine sextet. In the qNOR structure, the latter position is occupied by a (redox-insensitive) zinc atom, which most
likely is a crystallization artifact (Figure 3). A particular property of qNOR is the presence of an additional (14th) N-terminal
TMH that is followed by a long hydrophilic stretch of amino acids.
This sequence folds at the periplasmic site as a cyt c domain like
in cNOR, although a heme c itself is absent. Instead, the heme
c position is filled by a number of voluminous aromatic amino
acids. Two hydrophobic channels are observed in the structure
that run parallel to the membrane and connect the hydrophobic membrane interior with the active site. These channels might
function in substrate (NO) import and product (N2 O) export.
Two more features distinguish qNOR from cNOR: (1) the presence
of a quinol-binding site (Figure 4A) and of a water-filled channel
that likely plays a role in the supply of protons for NO reduction
(Eq. 4; Matsumoto et al., 2012; Shiro et al., 2012). The channel
leads from the bottom of the enzyme in the cytoplasm up to the
catalytic site.
The sequence comparison of the M. oxyfera and the other
unusual qNORs establish both resemblances and significant differences with respect to canonical qNORs. In DAMO_1889, all
characteristics are conserved, suggesting the protein to be a
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Frontiers in Microbiology | Evolutionary and Genomic Microbiology
genuine qNOR. Also in DAMO_2434, DAMO_2437, and their
relatives the overall folding is apparently maintained with respect
to the one of qNORs, as is inferred from sequence comparison
and structural modeling using qNOR of Geobacillus stearothermophilus (PDB 3AYF and 3AYG) as the template (not shown).
The arrangement of the 14 TMHs, the hydrophilic domain devoid
of heme c, all histidines except one, both putative substrate
channels and a portion of the amino acids related with the H+
channel are conserved. This suggests that DAMO_2434 and its
relatives, hereafter referred to as putative NOD, bind the electrontransferring heme b, the catalytic heme b3 , and non-heme iron
(or another catalytic metal). However, in the NODs one of the
coordinating histidines is consistently replaced by an asparagine
(Figure 4B). Similarly, a glutamate in close vicinity to the catalytic center, which has been implied with catalysis (Thorndycroft
et al., 2007; Flock et al., 2009; Hino et al., 2012; Shiro et al.,
2012) is substituted by a glutamine residue. Also the amino acids
lining the proposed H+ channel in qNOR have undergone several substitutions in the putative NODs. Most importantly, the
unusual “qNORs” lack a proper quinol-binding site. Conserved
residues that are assumed to constitute the quinol-binding site
in qNORs are substituted for amino acids that are unlikely to
provide a suitable site for quinol binding in the putative NODs
(Figure 4A). In summary, the latter apparently are unable of
accepting external electrons, they have a different catalytic site
and might be impeded in H+ uptake from outside the protein.
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reductases. The question then is what they do, presuming that
they do bear an important biological function – a reasonable
assumption given their high expression levels in M. oxyfera. It
is tempting to speculate that the modified proteins can bind
two NO molecules, rearrange N-O bonds with the aid of the
hemes and non-heme metal (iron or otherwise), and recombine both N and O atoms such that N2 and O2 are made. In
other words, the enzymes would act as an NO dismutase. At
this stage, this is speculation. The proof can only come from
the purification and rigorous characterization of these intriguing
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Received: 08 June 2012; paper pending
published: 27 June 2012; accepted: 10 July
2012; published online: 07 August 2012.
Citation: Ettwig KF, Speth DR, Reimann
J, Wu ML, Jetten MSM and Keltjens JT
(2012) Bacterial oxygen production in
the dark. Front. Microbio. 3:273. doi:
10.3389/fmicb.2012.00273
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Copyright © 2012 Ettwig, Speth,
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