Centre for Metalloprotein Spectroscopy and
Biology, School of Biological Sciences, University of East Anglia,
Norwich NR4 7TJ, United Kingdom
The bacterial nitric oxide reductase (NOR) is a divergent member of
the family of respiratory heme-copper oxidases. It differs from other
family members in that it contains an FeB-heme-Fe
dinuclear catalytic center rather than a CuB-heme-Fe
center and in that it does not pump protons. Several glutamate residues
are conserved in NORs but are absent in other heme-copper oxidases. To
facilitate mutagenesis-based studies of these residues in
Paracoccus denitrificans NOR, we developed two expression
systems that enable inactive or poorly active NOR to be expressed,
characterized in vivo, and purified. These are (i) a homologous system
utilizing the cycA promoter to drive aerobic expression of
NOR in P. denitrificans and (ii) a heterologous system
which provides the first example of the expression of an
integral-membrane cytochrome bc complex in
Escherichia coli. Alanine substitutions for three of the
conserved glutamate residues (E125, E198, and E202) were introduced
into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact
cells and membranes has demonstrated that two of the glutamates are
essential for normal levels of NOR activity: E125, which is predicted
to be on the periplasmic surface close to helix IV, and E198, which is
predicted to lie in the middle of transmembrane helix VI. The
subsequent purification and spectroscopic characterization of these
enzymes established that they are stable and have a wild-type cofactor
composition. Possible roles for these glutamates in proton uptake and
the chemistry of NO reduction at the active site are discussed.
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INTRODUCTION |
Many species of bacteria contain a
nitric oxide reductase (NOR) which catalyzes the reaction 2NO + 2e
+ 2H+ 
N2O + H2O (21, 26). The reduction of NO serves as a
key step in denitrification (in which N-oxyanions and N-oxides are used
as respiratory electron acceptors) and as a way of removing cytotoxic
NO. The NOR of Paracoccus denitrificans is an
integral-membrane protein that normally purifies as two-subunit complex
NorCB (10-12, 15). NorC is a monoheme membrane-anchored
c-type cytochrome. NorB is a divergent member of the family
of catalytic subunits from respiratory heme-copper oxidases (HCOs)
(21, 26). Typical features of catalytic subunits of the
HCOs are a core functional unit of 12 transmembrane helices, which bind
a magnetically isolated electron-transferring heme, and a dinuclear
active site, formed by a second heme magnetically coupled to a copper
ion (CuB). Seven conserved histidine residues, responsible
for ligating the three redox-active metal centers, can be identified in
helices II, VI, VII, and X. Each of these histidine residues is
conserved in the NorB subunit of NOR (21, 26, 29).
The key difference between the catalytic subunit of NOR and those of
other HCOs is the composition of the dinuclear center. In NorB there is
a nonheme iron (FeB) at the active site rather than copper
(CuB) (13), possibly because, under the highly
reducing conditions of the primordial biosphere, ferrous ions were more readily available than insoluble cuprous ions to the ancestral enzyme from which both NOR and HCOs evolved. Since it is likely that
denitrification preceded aerobic respiration in the biosphere, the
primary function of the ancestral oxidase was probably the reduction of
NO. Consequently, a key step in the evolution of aerobic life on earth
may have been the replacement of iron by copper in the ancestral
oxidase, allowing it to reduce oxygen more efficiently
(4). Recent biochemical studies have begun to reveal
additional differences in the catalytic pockets of NorB and HCOs. For
example, resonance Raman spectroscopy of the CO adduct of reduced NOR
has suggested that the catalytic pocket of NorB is more negatively
charged than those of HCOs (18). In addition, redox
potentiometry has indicated a midpoint potential of high-spin heme
b3 that is around 200 mV lower than that of high-spin heme a3 of cytochrome oxidase
(11). This may serve to prevent formation of a dead-end
Fe(II)-NO complex during the catalytic cycle.
High-resolution X-ray analysis of the crystal structures of cytochrome
c oxidase, together with site-directed mutagenesis, have led
to the identification of amino acid residues that are important in the
delivery of chemical and "pumped" protons from the cytoplasm to the
dinuclear center during the catalytic cycle and that define the
so-called K and D channels (1, 16, 32). The absence of
these residues from NOR suggests that the enzyme is not a proton pump
and that it takes the protons required for reduction of NO from the
periplasm, and there is experimental evidence consistent with this idea
(2, 3, 22). Hence, evolution of the ancestral NO-reducing
enzyme into an HCO involved the acquisition of not only a Cu-containing
dinuclear center but also a proton pumping mechanism. The primary
structures of NorB subunits reveal a number of conserved glutamic acid
residues in putative transmembrane helices and periplasmic loops, which
are absent in other HCOs (29). These are (P. denitrificans numbering) E122 in the helix III/IV loop, E125 at
the periplasmic surface of helix IV, E198 and E202, located one and two
helical turns, respectively, below the putative FeB ligand
(His194) in helix VI, and E267, located in the middle of helix VIII.
This sequence conservation, together with the energetic cost of placing
a charged residue in the lipid bilayer, suggests a functional
importance for these glutamates. Possible roles include FeB
binding, modulation of the charge of the catalytic pocket or of the
catalytic heme redox potential, and mediation of proton movements.
In order to investigate the role of conserved residues in bacterial
NORs, there has been a need to develop suitable expression systems for
catalytically inactive enzymes. Heterologous and homologous expression
systems would allow assessment of the physiological consequences of
mutations in NorB, as well as the production of pure enzyme for
structure-function studies. We have developed two such systems that
meet these criteria, and the characterization of enzymes with E125A,
E198A, and E202A substitutions in intact cells, membrane fractions, and
purified preparations is reported. The results demonstrate that E125
and E198 are not required for the assembly of a stable holo-NorCB
enzyme complex but have a critical role in NO reduction.
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MATERIALS AND METHODS |
Construction of a system for homologous expression of P. denitrificans norCB.
Plasmid pKPD1 is a clone of the entire
cycA (P. denitrificans cytochrome
c550) gene and promoter region in expression
vector pKK223-3, which has been modified to contain a unique
SalI site (25). A 325-bp
SalI-EcoRI fragment containing the
cycA promoter region was excised from pKPD1 and cloned into
pUC18 to yield pGB1. A 7.8-kb HindIII fragment
containing the norCBQDEF operon was excised from pEG8HI (a
gift from R. J. M. van Spanning, Vrije Universiteit,
Amsterdam, The Netherlands) and cloned into pUC18 to yield pNORHC.
There are two BsaBI restriction sites in the cycA
promoter region in pGB1. The first site cuts 1 bp downstream of the ATG
start codon of the cycA gene, and the second cuts 6 bp
downstream of the first. The norCBQDEF coding region, minus the norC promoter, was excised from pNORHC on a blunt-ended
5.7-kb SanDI-HindIII fragment and ligated
into pGB1 that had been digested with BsaBI. Clones
containing the 5.7-kb insert in the correct orientation were selected
and designated pCYCNOR1. The cycA-nor fusion was excised
from pCYCNOR1 on a 6.0-kb EcoRI-PstI fragment and
cloned into pBluescript KS+. Recombinant clones were designated pCYCNOR2. The 6.0-kb cycA-nor fusion was excised from
pCYCNOR2 by digestion with XbaI and HindIII
and cloned into broad-host-range vector pEG400 to yield pCYCNOR3,
generating a cycA-nor fusion that could be propagated in
P. denitrificans.
Construction of a norB::
-Km
mutant.
The 4-kb BglII fragment containing the
nor operon from pNORHC was cloned into the BamHI
site of pBluescript KS+ to yield pBg14. A 2.2-kb BamHI
-Km fragment was excised from pHP45-Km and cloned into the unique
BamHI site in pBg14, which is in norB, to
generate pBglkm. The insert from pBglkm was cloned as a 6.1-kb
XbaI-EcoRI fragment into pLITMUS28 to yield
pLitBglkm, which was then digested with XbaI and
SpeI, and the 6.1-kb fragment was cloned into the XbaI site of pUC18. A correctly oriented clone was selected,
and the plasmid was named pUCBglkm. These cloning steps resulted in the
location of the entire 6.1-kb DNA fragment containing the -Km
cassette and flanking DNA from the nor coding sequence on a
single EcoRI fragment. This fragment was introduced into the suicide vector pRVS1 to yield pRVSBglkm. Escherichia coli
S17.1 (pRVSBglkm) and P. denitrificans 1222 were then used
in biparental filter matings on L agar (16 h at 30°C). Cells were
removed from the filter by resuspension in L broth and plated onto L
agar (36 h at 37°C) containing rifampin, spectinomycin, kanamycin,
and X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside).
Streptomycin-sensitive, kanamycin-resistant white colonies were
designated P. denitrificans GB1 (genotype
norB::-Km). The presence of the
2.1-kb cartridge in norB was confirmed by direct genomic
PCR analysis.
Directed mutagenesis of norB.
pCYCNOR3 was digested
with XbaI, which cuts upstream of the cycA
promoter, and XhoI, which cuts in the middle of
norB. The 1.6-kb fragment (which contains the E125, E198,
and E202 codons) was ligated into
XbaI/XhoI-digested pBluescript KS+ to generate pNORXX16, which was transformed into E. coli DH5
. PCRs
were set up with complementary primer pairs suitable for introduction
of the E125A (GAA GCG), E198A (GAG GCC), E202A (GAG GCC),
and E198A plus E202A mutations. The following silent restriction sites were incorporated into the primers to allow for easy screening for
mutations by restriction digests: E125A, FspI; E198A,
SacI; E202A, ApaI; E198A plus E202A,
NaeI and ApaI. The template for PCR was pNORXX16,
and reactions were performed using the Quickchange (E198) or ExSite
mutagenesis kit (Stratagene). In the final stage of the protocol,
E. coli XL1-Blue transformed in each of the four ligation
reactions was spread onto L agar plates supplemented with 100 µg of
ampicillin/ml. Potential mutants were selected and sequenced to
establish their authenticity. The plasmids carrying codons leading to
the E125A, E198A, E202A, and E198A plus E202A mutations were designated
pNOR125A, pNOR198A, pNOR202A, and pNOR198202A, respectively. The 1.6-kb
XbaI-XhoI fragments from the four mutant plasmids
were cloned into XbaI/XhoI-digested pCYCNOR. The
resulting plasmids were then digested with FspI (E125A),
SacI (E198A), or ApaI (E202A and E198A plus
E202A), as appropriate. All clones were found to have the expected
restriction pattern. These plasmids were designated p125CNOR, p198CNOR,
p202CNOR, and p198202CNOR and were introduced into P. denitrificans GB1 by triparental matings with the corresponding
E. coli DH5 transformants and E. coli JM83
harboring helper plasmid pRK2013.
Construction of pNOREX used for the expression of the
norCBQDEF operon in E. coli.
The 6.0-kb
XbaI-HindIII fragment, containing the
cycA-nor fusion from pCYCNOR3, was cloned into pUC18 to
yield pNOREX, which has the nor operon in the correct
orientation to allow expression from the lac promoter.
pNOREX was found to be unstable in E. coli DH5, so strain
JM109 (lacIq), in which pNOREX was more stable,
was used as the host. Similar procedures were used to construct p125EX,
p198EX, p202EX, and p198202EX, using the 6.0-kb
XbaI-HindIII fragments from p125CNOR, p198CNOR, p202CNOR, and p198202CNOR.
Anaerobic growth of P. denitrificans.
P.
denitrificans strains were grown aerobically at 37°C in 50 ml of
L broth supplemented with the appropriate antibiotics. For each strain,
a 500-ml bottle of succinate-nitrate minimal medium, supplemented with
the appropriate antibiotics, was inoculated with a 1% volume from the
L broth cultures. The bottle was then sealed, and the cells were mixed
thoroughly by inversion. The starting optical density at 610 nm
(OD610) was determined, and, to avoid further introduction
of oxygen during anaerobic growth, the contents of the inoculated
bottles were aliquoted into 25-ml bottles. Each 25-ml bottle was then
tightly sealed and incubated at 30°C. A single bottle was opened for
each time point in the growth curve and the OD610 was
recorded. Also, at each time point, 1.5 ml of cells was centrifuged for
5 min at 13,000 rpm in a bench top microcentrifuge. The culture
supernatant was then assayed colorimetrically for nitrite.
Analytical methodologies.
NO reductase activity was measured
amperometrically using a Clark-type electrode, essentially as
previously described (10, 13), but using ascorbate,
phenazine methosulfate, and horse heart cytochrome c as the
electron donor/mediator system. Cytochrome oxidase activity was
measured spectrophotometrically by monitoring the NOR-dependent
oxidation kinetics of reduced horse heart cytochrome c in
aerated cuvettes. Protein levels were estimated using the bicinchoninic
acid method with bovine serum albumin as a standard. The stain for
heme-linked peroxidase activity, which is specific for
c-type cytochromes, was as previously described
(20). The rates given in Tables 2 and 3 are representative
data taken from samples prepared from at least two independent cultures.
EPR, UV-Vis, and mediated redox potentiometry.
Electron
paramagnetic resonance (EPR) spectra were recorded using an ER-200D
X-band spectrometer (Bruker Spectrospin) interfaced to an ESP1600
computer and fitted with a liquid-helium flow cryostat (ESR-9; Oxford
Instruments). UV-visible (UV-Vis) spectra were collected using an
Aminco SLM DW2000 spectrophotometer. Samples for UV-Vis spectra and
redox titrations were at 25°C in 50 mM Tris-HCl (pH 7.5). Mediated
redox potentiometry was performed as previously described
(11). Dithionite and ferricyanide were used as the
reductant and oxidant, respectively. Redox mediators were phenazine
methosulfate, phenazine ethosulfate, diaminodurene, 4-hydroxynaphthoquinone, 5-anthraquinone 2-sulfonate, 6-anthraquinone 2,6-disulfonate, and benzyl viologen (at a final concentration of 20 µM). Quinhydrone was used as a redox standard
(Em,7.0 = +295 mV). All potentials quoted are with
respect to that of the normal hydrogen electrode. Redox titrations were
fitted using a customized program in table-curve 2D (Jandel Scientific)
allowing estimates of Em and multiple independent
n = 1 components to float as appropriate
(11). The error for each Em was estimated from multiple titrations to be ±20 mV.
Purification of NOR.
Recombinant NOR was purified from
15-liter cultures of E. coli grown in L broth in an aerated
bioreactor. When the culture density reached an OD650 of
0.4, the culture was induced for 4 h with 1 mM IPTG
(isopropyl--D-thiogalactopyranoside). Cells were then
harvested by cross-flow filtration and broken in a French press.
Membranes were recovered by centrifugation and suspended in 100 mM
Tris-HCl (pH 7.6)-50 mM NaCl-1 mM EDTA, sonicated, and solubilized in
1% (wt/vol) n-dodecyl--D-maltoside (4°C
for 1 h). The sample was then centrifuged at 45,000 rpm in a
Beckman 70Ti rotor for 1 h at 4°C. The supernatant was
immediately diluted 10-fold with buffer to avoid precipitation of the
protein. The protein sample was then purified using Q-Sepharose (0 to
500 mM NaCl gradient) and Cu-IMAC (2.5 to 50 mM imidazole gradient)
chromatographies, as previously described (13). The
elution buffer was 50 mM Tris-HCl (pH 7.5)-0.1% dodecyl maltoside.
Fractions containing NOR were identified spectroscopically.
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RESULTS AND DISCUSSION |
Characterization of engineered NOR in intact cells and
membrane fractions of P. denitrificans.
The utility of
pCYCNOR3 (Table 1) for
nor expression in P. denitrificans depends on its
ability to express norCB under growth conditions for which
NOR is not essential. Thus, the expression of an engineered
norCB that encodes an inactive or poorly active NOR is
possible. The nor promoter is only active under anaerobic conditions and is dependent on the presence of NO (14, 17, 27). However, in pCYCNOR3 the nor genes are
transcribed from the P. denitrificans cycA promoter (from
the cytochrome c550 gene), which is known to be
active under some aerobic growth conditions (20, 25).
P. denitrificans strains GB1
(norB::) and GB1(pCYCNOR3) were grown under a
range of conditions, and the NOR activities of the membrane fractions
were determined. GB1 displayed no detectable NOR activity under any of
the growth conditions tested (Table 2).
By contrast, NOR activity was detectable in GB1(pCYCNOR3) under all
growth conditions tested. Activity was lowest following aerobic growth
on succinate medium and was an order of magnitude higher in membranes
prepared from cells grown aerobically on methylamine, anaerobically on
succinate-nitrate medium, or aerobically to late stationary phase on L
broth (Table 2).
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TABLE 2.
NOR activities of membranes prepared from P. denitrificans GB1 grown under different conditions with
different plasmids
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These expression studies demonstrated that the cycA promoter
is capable of driving expression of the nor operon under
growth conditions for which NOR is nonessential. Having established
this, mutations leading to E125A, E198A, E202A, and E198A plus E202A substitutions were introduced into the norB gene (Table 1)
and the engineered enzymes were expressed in aerobic methylamine-grown cultures of GB1 (norB::). There was no
detectable NOR activity in membranes from the strains expressing
enzymes with the E125A, E198A, and E198A plus E202A substitutions and
intermediate levels of activity in membranes from the strain expressing
NOR with the E202A substitution (Table 2). Immunochemistry was employed
to assess the expression of the catalytic NorB subunit. Membrane fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) alongside a sample of purified NOR. The gel
was Western blotted and then probed with an anti-NorB antibody. A
strongly reactive band that corresponded to NorB could be observed in
the lane containing purified NOR. This protein was absent from
norB:: mutant GB1 but present in GB1(pCYCNOR3) (Fig. 1A). The same band was also present
in GB1(p125CNOR), GB1(p198CNOR), GB1(p202CNOR), and GB1(p198202CNOR).
Although there was some sample-to-sample heterogeneity, analysis of
three independent membrane preparations suggested that levels of
expression of the NorB polypeptide in all of the strains carrying
either the wild-type or mutant forms of the norB gene were
similar. This confirmed that NorB biosynthesis and stability were
similar for all of the engineered enzymes and so could not account for
the pronounced differences in NOR activity observed in membranes
expressing these enzymes.
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FIG. 1.
Heme-stained SDS-PAGE gel and anti-NorB-probed Western
blot of membrane fractions from P. denitrificans and
E. coli. (A) Anti-NorB-probed Western blot of P. denitrificans membranes. Membranes were prepared from cells grown
aerobically on L broth and solubilized in 1% dodecyl maltoside.
Fifteen microliters (5 to 10 µg of protein) of each sample was loaded
onto the SDS-PAGE gel, which was subsequently used for the Western
blotting. Lane 1, purified NorCB; lane 2, strain 1222; lane 3, GB1;
lane 4, GB1(pCYCNOR3); lane 5, GB1(p125CNOR); lane 6, GB1(p198CNOR);
lane 7, GB1(p202CNOR); lane 8, GB1(p198202CNOR). (B) Heme-stained gel
of E. coli membranes. Lane 1, JM109; lane 2, JM109(pEC86);
lane 3, JM109(pNOREX, pEC86); lane 4, JM109(p125EX, pEC86); lane 5, JM109(p198EX, pEC86); lane 6, JM109(p202EX, pEC86); lane 7, JM109(p198202EX, pEC86). (C) Anti-NorB-probed Western blot of E. coli membranes. Lane 1, JM109; lane 2, JM109(pEC86); lane 3, JM109(pNOREX, pEC86); lane 4, JM109(p125EX, pEC86); lane 5, JM109(p198EX, pEC86); lane 6, JM109(p202EX, pEC86); lane 7, JM109(p198202EX, pEC86); lane 8, purified NorCB. Membranes were
solubilized in 1% dodecyl maltoside, and 15 µl (5 to 10 µg of
protein) was loaded into each well of the SDS-PAGE gels.
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To assess the physiological competence of the engineered NORs, P. denitrificans 1222, GB1, GB1(pCYCNOR3), GB1(p125CNOR),
GB1(p198CNOR), and GB1(p202CNOR) were cultured under anaerobic
denitrifying conditions with succinate as the carbon source, ammonium
as the nitrogen source, and nitrate as a respiratory electron acceptor
(Fig. 2A). During the first 6 h of
anaerobic incubation, strains 1222 (wild type) and GB1
(norB::) exhibited similar growth kinetics.
This growth period was accompanied by a rapid accumulation of nitrite in the culture supernatant, which could be attributed to the
respiratory reduction of nitrate to nitrite (Fig. 2B). After this
period, growth of GB1 was almost completely attenuated (Fig. 2A),
although a net accumulation of nitrite continued throughout the 20-h
duration of the growth experiment (Fig. 1B). It is likely that after
6 h some of the nitrite initially produced by the culture was
reduced to NO by the cytochrome cd1 nitrite
reductase. The NO cannot be further reduced in the absence of NOR and
so inhibits growth. Introduction of the nor-expressing clone
pCYCNOR3 into GB1 restored the wild-type capacity for anaerobic
denitrifying growth (Fig. 2A). Nitrogen gas bubbles could be observed
during growth of both 1222 and GB1(pCYCNOR3), indicative of complete
denitrification. However, there were significant differences in the
nitrite extrusion profiles during growth of strains 1222 and
GB1(pCYCNOR3). The wild-type strain accumulated nitrite in the growth
medium throughout growth, but in GB1(pCYCNOR3) the nitrite reached a
steady concentration (7 to 12 mM) between 6 and 18 h, increasing
rapidly again thereafter (Fig. 2B). These differences may be a
consequence of expressing nor from the cycA
promoter. The nor promoter is coregulated with the nitrite
reductase genes by the NO-responsive activator NNR (14, 17, 27,
29); this coordinate regulation is lost in the recombinant
expression system.
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FIG. 2.
Growth curves of P. denitrificans 1222 ( ),
GB1 ( ), GB1(pCYCNOR3) ( ), GB1(p125CNOR) ( ), GB1(p198CNOR)
( ), and GB1(p202CNOR) ( ). Cultures were grown under anaerobic
denitrifying conditions. (A) Growth kinetics, monitored via
OD610, of 1222, GB1, and GB1(pCYCNOR3). (B) Nitrite
accumulation kinetics during growth of 1222, GB1, and GB1(pCYCNOR3).
(C) Growth kinetics, monitored via OD610, of GB1(p125CNOR),
GB1(p198CNOR), and GB1(p202CNOR). (D) Nitrite accumulation kinetics
during growth of GB1(p125CNOR), GB1(p198CNOR), and GB1(p202CNOR).
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Strains GB1(p125CNOR) and GB1(p198CNOR) resembled GB1 in that they were
able to grow during the first 6 h after inoculation (Fig. 2C) by
virtue of the energy-conserving reduction of nitrate to nitrite, which
accumulated in the culture supernatant (Fig. 2D). Thereafter, no
further growth was apparent, presumably as a result of the failure to
reduce the NO derived from nitrite reduction at sufficiently rapid
rates to prevent toxicity. GB1 expressing NORE202A showed
almost complete complementation (Fig. 2C). In all three cases the
growth phenotypes reflect the relative levels of NOR activity observed
in membrane fractions prepared from the methylamine-grown cells (Table
2).
Characterization of engineered P. denitrificans NOR in
intact cells and subcellular fractions of E. coli.
Homologous expression of norCB proved essential for
assessing the physiological competence of engineered NORs and for
establishing that they were synthesized and stable. To provide large
quantities of NOR for purification and spectroscopic analysis, a
heterologous nor expression system utilizing the
IPTG-inducible lacZ promoter of pUC18 in E. coli
JM109 was developed. To facilitate expression, E. coli
was cotransformed with pNOREX (Table 1) and pEC86, which contains
the cytochrome c assembly (ccm) genes (19,
22). Dithionite-reduced UV-Vis spectra revealed differences
between JM109, JM109(pEC86), and JM109(pEC86/pNOREX) membrane extracts.
The reduced spectrum of detergent-solubilized membranes from JM109
shows a peak at approximately 560 nm typical of E. coli
respiratory complexes containing b-type heme, such as the
cytochrome bo3 oxidase and the formate
dehydrogenase (Fig. 3). There is no
indication of the presence of any c-type cytochromes, since
there is no absorption peak at around 550 nm. The JM109(pEC86) extract
shows an overall increase in the intensity of the spectrum and a slight
shift of the peak towards 558 nm. This is probably due to the
expression of the CcmE protein (from pEC86), which is known to absorb
maximally at 558 nm when in the reduced form (22). The
clearest spectral changes occurred in JM109(pEC86/pNOREX), where two
peaks with almost equal intensities at approximately 550 and 558 nm can
be resolved (Fig. 3). The peak at 550 nm is indicative of the presence of a c-type cytochrome and is likely to arise from NorC. The
intensity at 558 nm is likely to arise from a combination of CcmE,
various respiratory complexes, and also the b hemes of NorB.
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FIG. 3.
UV-Vis absorption spectra of membranes prepared from
E. coli JM109, JM109(pEC86), and JM109(pEC86/pNOREX). The
strains were grown aerobically in 500-ml Luria-Bertani medium (in
2.5-liter baffled flasks) and induced at an OD610 of 0.4 with 1 mM IPTG. Cells were harvested 4 h after induction, and
membranes were prepared as described in Materials and Methods. Spectra
were acquired from suspensions of 100 µg of membranes per ml.
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Detergent-solubilized membrane extracts of JM109, JM109(pEC86), and
JM109(pEC86/pNOREX) were subjected to SDS-PAGE, and the gel was stained
for heme-dependent peroxidase activity to enable detection of
c-type cytochromes. The JM109 membrane extract shows very
little covalently attached heme (Fig. 1B). The faint
high-molecular-weight bands are thought to arise from noncovalently
bound b heme, which had not fully dissociated from some of
the respiratory complexes. The JM109(pEC86) membrane extract contained
a band at 19 kDa, which stained strongly for heme and which is likely
to be CcmE (19, 22). In addition this extract contained a
30-kDa heme-staining polypeptide which is likely to arise from an
endogenous E. coli protein, since it is also present in the
JM109 membrane extract, albeit at extremely low levels. The membrane
extract from cells containing both pEC86 and pNOREX has an additional
18-kDa heme-staining polypeptide that migrated slightly faster than the
CcmE polypeptide. The molecular mass of this heme-staining polypeptide
is consistent with it being NorC. To confirm the presence of NorB, all
three membrane extracts resolved by SDS-PAGE were Western-blotted and probed with an anti-P. denitrificans NorB antibody (Fig.
1C). There was no cross-reacting band in the lanes loaded with JM109 and JM109(pEC86) extracts. However, a single strongly cross-reacting band that migrated to the same position as the NorB polypeptide of
purified NOR could be clearly identified in the JM109(pEC86/pNOREX) extract. In agreement with the apparent expression pattern of NorC and
NorB from the heme-staining and immunochemical analysis, membranes
prepared from JM109 and JM109(pEC86) both displayed no detectable NOR
activity, whereas JM109(pEC86/pNOREX) membranes displayed significant
activity (Table 3). These data confirmed that P. denitrificans NOR was expressed and active in
E. coli JM109 containing pNOREX and the ccm
coexpression plasmid pEC86. This represents the first example of the
expression of a large integral-membrane respiratory cytochrome
bc complex in E. coli. The coexpression of the
ccm genes from pEC86 was critical to the success of this
strategy since the NorC subunit did not assemble efficiently in its
absence (data not shown). It should also be noted that pNOREX contained
the whole norCBQDEF operon. Attempts to express NOR in the
absence of the norQDEF genes were unsuccessful, but a
systematic study of the role of each of these genes in the assembly
and/or stability of NOR was not undertaken at this stage.
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TABLE 3.
NOR and oxidase activities in membranes prepared from
E. coli JM109 expressing wild-type or mutant forms of NOR
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To assess the activity of the engineered NORs in E. coli,
p125EX, p198EX, p202EX, and p198202EX were all introduced into JM109 with pEC86. Solubilized membrane extracts were subjected to SDS-PAGE, and the gel was stained for covalently bound heme (Fig. 1B). The 18-kDa
polypeptide identified as NorC was present in all of the extracts. The
presence of NorB was confirmed using the NorB antibodies (Fig. 1C).
Membrane extracts of the E. coli strains expressing engineered NorB were assayed for NOR activity (Table 3). The activities
followed a pattern similar to those obtained for the mutant enzymes
expressed in P. denitrificans; no activity was detected for
the E198A mutant, and the E202A mutant had the highest activity. The
only major discrepancy was that the E125A mutant had no detectable NOR
activity when expressed in P. denitrificans but did show a
very low (5% of wild-type) activity when expressed in E. coli. This residual activity may have been too low to detect in
P. denitrificans as a consequence of the background NOR
activity of cytochrome oxidases.
It was also possible to determine whether the P. denitrificans NOR expressed in E. coli possessed an
oxidase activity. This assay had previously only been possible with
purified enzyme from P. denitrificans, because of the high
levels of cytochrome c oxidase activity that are present in
P. denitrificans membranes. E. coli, however,
contains only quinol oxidases. Low levels of cytochrome c
oxidase activity were detected in JM109(pNOREX, pEC86) (Table 3). There
was no activity in cells that did not harbor pNOREX, confirming that
the activities detected were due to NOR. Significantly, no cytochrome
oxidase activity could be detected in NORs with the E125A or E198A substitution.
UV-Vis and EPR characterization of purified preparations of
NORREC, NORE125A, and
NORE198A.
Recombinant NOR (NORREC),
NORE125A, and NORE198A were purified from
15-liter L broth cultures of E. coli JM109 carrying the
appropriate plasmid. The key step in the purification (described in
Materials and Methods) involved the Q-Sepharose column from which NorCB eluted in two cytochrome-containing peaks at around 320 and 450 mM NaCl
(Fig. 4). The first of these also
contained large amounts of CcmE. In the second peak, NorCB was
separated from other contaminating cytochromes and proteins. The NOR
from this second peak was collected and separated on the IMAC column
prior to characterization. The ratio of the two elution peaks from the
Q-Sepharose column varied from preparation to preparation and
influenced the final yield of purified protein, which was 5 to 10 mg
per 15 liters of culture.
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FIG. 4.
The elution profile of NorCB from a Q-Sepharose column.
Fraction volumes were 12 ml. A 0 to 200 mM salt gradient was run for
the first 120 ml. This was then switched to a 200 to 500 mM gradient
for the following 720 ml. , cytochrome absorbance at 410 nm; ,
protein absorbance at 280 nm.
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The patterns of NOR and oxidase activities in purified
NORREC and NORE125A and NORE198A
reflected those observed in membrane fractions. The turnover number
for NORREC was comparable to that of native NOR
(purified from P. denitrificans; NORNAT)
determined in side-by-side experiments and was in the range of 40 to 70 electrons s1 for NO reduction and 2 to 5 electrons
s1 for oxygen reduction. NORE198A had no
detectable activity, and NORE125A had a low turnover number
in the range of 3 to 5 electrons s1 for NO reduction and
around 1 electron s1 for oxygen reduction. Comparison of
the UV-Vis spectra of the oxidized forms of NORNAT,
NORREC, NORE125A, and NORE198A
revealed absorption features typical of the Soret band (411 nm) and
absorption bands (520 to 570 nm) of low-spin ferric hemes. On
reduction with dithionite, the Soret band shifted to 420 nm and an
increase in absorption at 550 and 560 nm characteristic of the bands of low-spin ferrous c and b hemes,
respectively, was observed. These features were essentially identical
for all four enzymes, and representative spectra for NORREC
are shown in Fig. 5A.
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FIG. 5.
Visible absorption spectra and redox potentiometry of
NORE198. (A) "Air-oxidized" (thin line) and
"dithionite-reduced" (thick line) absorption spectra. (B) Redox
titration of NORREC monitored at 550 to 700 nm. (C) Redox
titration of NORREC monitored at 560 to 700 nm. (D) Redox
titration of NORREC monitored at 606 to 700 nm. The solid
curves (B to D) show fits with n = 1 Nernstian curves
using midpoint potentials of +316, +366, and +32 mV, respectively. All
spectra and titrations were performed on samples incubated at 20°C in
20 mM Tris-HCl (pH 7.5)-0.02% dodecyl maltoside-340 mM NaCl-0.5 mM
EDTA.
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A major difference in the spectra of the four enzymes was observed in a
charge transfer (CT) band at around 600 nm, which arises from the
high-spin heme b3 of the dinuclear center
(11, 13). This CT band disappears on reduction of the
enzyme, enabling it to be resolved most clearly in "oxidized minus
reduced" spectra (Fig. 6). In
NORNAT the wavelength for maximum absorbance
(
max) is 595 nm (Fig. 6A and E), in NORREC
(Fig. 6B and F) and NORE125A (Fig. 6D and H) it is red
shifted to 606 nm, and in NORE198A (Fig. 6C and G) it is a
mixture of the 595- and 606-nm forms. This CT band has been seen in the
visible absorption spectrum of NOR in a number of published
preparations, but variations in its position and intensity have been
noted (8, 10, 11, 13, 15), and it is barely visible at all
in enzymes from Pseudomonas stutzeri (12, 15).
The position of this CT band cannot be correlated with differences in
enzyme activity, and its variable max probably arises
from differences in the coordination environment of the high-spin heme
b of the resting enzymes. The precise nature of these
differences cannot be resolved at present, but is likely to involve the
sixth coordination position of the ferric heme iron in the
resting-state enzymes. Significantly, this difference in the resting
state of the enzymes cannot account for the low activity of
NORE125A and NORE198A, since NORNAT
("595" species) and NORREC ("606" species) both
exhibit high enzymatic activity. The differences in resting states of
the NOR enzymes are reminiscent of those of the E. coli
cytochrome bo quinol oxidase, in which the active-site dinuclear center can exhibit considerable heterogeneity (30, 31).
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FIG. 6.
Absorption spectra of NORNAT,
NORREC, NORE198A, and NORE125A
showing the CT band arising from high-spin heme
b3. (A to D) Air oxidized spectra of
NORNAT (A), NORREC (B), NORE198A
(C), and NORE125A (D). (E to H) Oxidized-minus-reduced
difference spectra (thick lines) of NORNAT (E),
NORREC (F), NORE198A (G), and
NORE125A (H) and three-electron-reduced minus
four-electron-reduced difference spectra (thin lines) of
NORNAT (E), NORREC (F), NORE198A
(G), and NORE125A (H). These were obtained by subtracting
spectra collected at around  50 mV from spectra collected at around
+140 mV.
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NORNAT, NORREC, NORE125A, and
NORE198A were also examined by EPR spectroscopy. The
EPR spectrum of NORNAT shows the presence of two
low-spin (s = 1/2) ferric hemes (Fig. 7A), one with a typical rhombic spectrum
(gz = 3.00, gy = 2.25, gx = 1.46) and the other with a high
gmax signal (gz = 3.55). These signals have previously been ascribed to the low-spin
bis-His-coordinated heme b and the low-spin
His-Met-coordinated heme c of NOR and yield spin
quantitations of 1:1 (5). Both signals could also be
resolved in NORREC and were present at relative intensities
similar to those of NORNAT (Fig. 7B). The major difference
between NORNAT and NORREC was in the signals at
g 6 and g 4.2. The signal at
g 6 arises from s = 
high-spin ferric heme, most likely a small proportion of the heme
b from the dinuclear center that is not magnetically coupled
to the nonheme iron. The small increase of this uncoupled population of
the dinuclear center in NORREC is also reflected by an
increase in the structured g 4.2 resonance that
arises from the uncoupled FeB nonheme iron. The EPR spectra of NORE125A and NORE198A were essentially
identical to that of the NORREC enzyme (not shown). Given
that NORREC is fully active, the increased population of
the non-magnetically coupled dinuclear center compared to that for
NORNAT cannot account for the low activity of the
NORE125A and NORE198A enzymes.
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FIG. 7.
X-band EPR spectra of air-oxidized native (A) and
recombinant wild-type (B) NOR. The spectra were recorded at 10 K, 9.44 GHz, and 2 mW microwave power. The feature at ca. 2.01 in spectrum A
may arise from a small (<1%) contaminant of a [3Fe4S]1+
center from the P. denitrificans membrane-bound nitrate
reductase.
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Spectropotentiometric characterization of NORREC,
NORE125A, and NORE198A.
Visible
absorption spectra of NORNAT, NORREC,
NORE125A, and NORE198A were collected at a
number of defined redox potentials. In all cases, increases in the
intensities of the bands of the low-spin c heme (550 nm)
and low-spin b heme (560 nm) were observed between ca. +400
and +200 mV. The absorption differences at 550 to 700 nm and 560 to 700 nm over this potential range were plotted as a function of redox
potential and the midpoint potentials were derived by fitting single
component n = 1 Nernstian curves to the data (a
representative data set for NORREC is shown in Fig. 5B and
C). For all four enzymes, the midpoint potentials of the low-spin
c heme lay in the range of +310 to +322 mV (Table
4). Those of the low-spin b
heme were slightly higher, in the range +345 to +401 mV (Table 4).
These values are all consistent with these low-spin heme centers
mediating electron transfer from the physiological electron donors,
periplasmic cytochrome c550 (Em = +265 mV) and pseudoazurin (Em = +230 mV), to the
dinuclear center.
Analysis of the 595/606-nm CT band arising from high-spin heme
b is more complex. We have previously reported that the
max of this CT band in NORNAT is shifted
from 595 to 606 nm following reduction of the low-spin c and
b hemes and FeB to yield the
"three-electron-reduced" enzyme (11). We have argued
that this shift in max reflects a change in the
coordination environment of high-spin heme b that
accompanies the reduction of the spectroscopically silent
FeB (Em = +320 mV) (11). This
idea is supported by the present study, and an illustrative absorption
spectrum of the three-electron-reduced form collected at +140 mV is
presented in Fig. 6E. This reveals that the extinction coefficient of
the 595-nm CT band is around fivefold greater (approximately 6 mM1 cm1) than that of the 606-nm CT band
(approximately 1.2 mM1 cm1).
In the light of these data, consideration of the oxidized spectrum of
NORE198A (Fig. 6G) suggests that around 20% of the enzyme
is in the 595-nm form and around 80% is in the 606-nm form. The
spectrum is largely unchanged when the Eh is lowered to
+250 mV (not shown). However, lowering the Eh from +250 to
+140 mV results in the loss of the 595-nm feature of the
NORE198A spectrum and a 20% increase in the intensity of
the 605-nm band (Fig. 6G). This increase can be ascribed to the
reduction of FeB in the 20% of the enzyme population that
is in the 595-nm form. The absorption changes are too small to allow
the plotting of a Nernstian curve, but the data place the
Em of NORE198A FeB at around +200
mV, considerably lower than that of the FeB in
NORNAT (+320 mV) (11). Both NORREC
and NORE125A are in an almost homogenous 606-nm form in the
fully oxidized enzyme, and reduction to +140 mV does not significantly
change the form of this CT band (Fig. 6F and H). Thus, consideration of
NORNAT, NORREC, NORE125A, and
NORE198A, poised at around +140 mV, reveals that all four
enzymes are in a similar spectroscopic state in which it is likely that
low-spin hemes c and b and FeB are
reduced and high-spin heme b remains oxidized in a 606-nm
state. The 606-nm bands of NORNAT, NORREC,
NORE125A, and NORE198A all disappear as the
Eh is lowered from +140 mV to 80 mV. This reflects the
reduction of high-spin heme b3 to yield the
fully (four-electron-reduced) enzyme. The reduction of this center can be fitted to single n = 1 Nernstian curves (Fig. 5D;
shown for NORREC only) which yield midpoint redox
potentials that lie in the range of +2 to +40 mV for all four forms of
NOR studied (Table 4). We have previously argued that the low potential
of high-spin heme b3 provides a thermodynamic
barrier that prevents reduction of this site during the catalytic cycle
(11). If heme b3 becomes reduced,
then, potentially, a dead-end ferrous nitrosyl complex could form. This
thermodynamic barrier would exist in each of the four enzyme types
discussed in this paper, since in each case the difference in reduction
potential between low-spin heme b and the active-site heme
is at least 300 mV.
In conclusion, the accumulated spectroscopic data for purified
NORNAT, NORREC, NORE125A, and
NORE198A demonstrate that the inactivity of
NORE125A and NORE198A is unlikely to be
accounted for by either enzyme instability, failure to insert
cofactors, or perturbation of the redox potentials of the heme
cofactors. The location of E198 one helical turn below a likely
FeB ligand (H194) in helix VI strongly implicates it as
contributing to the immediate environment of FeB, which is also consistent with the preliminary suggestion that the Em
of the FeB is perturbed in NORE198A. Certainly
FeB, which unlike CuB prefers an octahedral
coordination environment, is likely to have at least one extra
protein-derived ligand. However, E198 is also well placed to serve as a
base for NO radical chemistry or for delivery of catalytic protons.
More-detailed spectroscopic and electrochemical studies on
NORE198A will now be undertaken to explore these
possibilities. The importance of E125 is perhaps most surprising given
that it is located towards the periplasmic face of helix IV and is not
predicted to be close to the FeB. Previous studies have
indicated that NOR is not proton translocating (2, 3, 23)
and that the two chemical protons required for NO reduction are taken
up from the periplasm. Given the conservation of E125, a role in proton
uptake should be considered.
We are grateful to James Moir for valuable ideas at the outset of
this work; Lola Roldán, Andrew Thomson, and Myles Cheeseman for
discussion and help in strategy development; Karen Grönberg and
Jeremy Thornton for provision of native NOR; Matti Saraste for
provision of antibody to NorB; Werner Klipp, Rob van Spanning, and
Lynda Thöney-Meyer for provision of strains and plasmids; and
Adam Baker and Louise Prior for collecting the EPR spectra.
The work was funded by BBSRC grant 83/C10160, the award of a
BBSRC/EPSRC special studentship to G.B., the UEA innovation fund, and
CEC grant EC BIO-CT98-0507. N.J.W. is a Wellcome Trust University Award
Lecturer (054798/Z/98Z).
| 1.
|
Adelroth, P.,
R. B. Gennis, and P. Brzezinski.
1998.
Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides.
Biochemistry
37:2470-2476[CrossRef][Medline].
|
| 2.
|
Bell, L. C.,
D. J. Richardson, and S. J. Ferguson.
1992.
Identification of nitric oxide reductase activity in Rhodobacter capsulatus: the electron transport pathway can either use or bypass both cytochrome c2 and the cytochrome bc1 complex.
J. Gen. Microbiol.
138:437-443[Medline].
|
| 3.
|
Carr, G. J.,
M. D. Page, and S. J. Ferguson.
1989.
The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification.
Eur. J. Biochem.
179:683-692[Medline].
|
| 4.
|
Castresana, J., and M. Saraste.
1995.
Evolution of energetic metabolism: the respiration-early hypothesis.
Trends Biochem. Sci.
20:443-448[CrossRef][Medline].
|
| 5.
|
Cheesman, M. R.,
W. G. Zumft, and A. J. Thomson.
1998.
The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases.
Biochemistry
37:3994-4000[CrossRef][Medline].
|
| 6.
|
de Vries, G. E.,
N. Harms,
J. Hoogendijk, and A. H. Stouthamer.
1989.
Isolation and characterisation of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA modifying property.
Arch. Microbiol.
152:52-57[CrossRef].
|
| 7.
|
Fellay, R.,
J. Frey, and H. Krisch.
1987.
Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria.
Gene
52:147-154[CrossRef][Medline].
|
| 8.
|
Fujiwara, T., and Y. Fukumori.
1996.
Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.
J. Bacteriol.
178:1866-1871[Abstract/Free Full Text].
|
| 9.
|
Gerhus, E.,
P. Steinrucke, and B. Ludwig.
1990.
Paracoccus denitrificans cytochrome c1 gene replacement mutants.
J. Bacteriol.
172:2392-2400[Abstract/Free Full Text].
|
| 10.
|
Girsch, P., and S. de Vries.
1997.
Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans.
Biochim. Biophys. Acta
1318:202-216[Medline].
|
| 11.
|
Grönberg, K. L.,
M. D. Roldán,
L. Prior,
G. Butland,
M. R. Cheesman,
D. J. Richardson,
S. Spiro,
A. J. Thomson, and N. J. Watmough.
1999.
A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.
Biochemistry
38:13780-13786[CrossRef][Medline].
|
| 12.
|
Heiss, B.,
K. Frunzke, and W. G. Zumft.
1989.
Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri.
J. Bacteriol.
171:3288-3297[Abstract/Free Full Text].
|
| 13.
|
Hendriks, J.,
A. Warne,
U. Gohlke,
T. Haltia,
C. Ludovici,
M. Lubben, and M. Saraste.
1998.
The active site of the bacterial nitric oxide reductase is a dinuclear iron center.
Biochemistry
37:13102-13109[CrossRef][Medline].
|
| 14.
|
Hutchings, M. I., and S. Spiro.
2000.
The nitric oxide regulated nor promoter of Paracoccus denitrificans.
Microbiology
146:2635-2641[Abstract/Free Full Text].
|
| 15.
|
Kastrau, D. H.,
B. Heiss,
P. M. Kroneck, and W. G. Zumft.
1994.
Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties.
Eur. J. Biochem.
222:293-303[Medline].
|
| 16.
|
Konstantinov, A. A.,
S. Siletsky,
D. Mitchell,
A. Kaulen, and R. B. Gennis.
1997.
The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer.
Proc. Natl. Acad. Sci. USA
94:9085-9090[Abstract/Free Full Text].
|
| 17.
|
Kwiatkowski, A. V.,
W. P. Laratta,
A. Toffanin, and J. P. Shapleigh.
1997.
Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.
J. Bacteriol.
179:5618-5620[Abstract/Free Full Text].
|
| 18.
|
Moenne Loccoz, P., and S. deVries.
1998.
Structural characterisation of the catalytic high spin heme b of nitric oxide reductase.
J. Am. Chem. Soc.
120:5147-5152[CrossRef].
|
| 19.
|
Reincke, B.,
L. Thony-Meyer,
C. Dannehl,
A. Odenwald,
M. Aidim,
H. Witt,
H. Ruterjans, and B. Ludwig.
1999.
Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.
Biochim. Biophys. Acta
1411:114-120[Medline].
|
| 20.
|
Roldan, M. D.,
H. J. Sears,
M. R. Cheesman,
S. J. Ferguson,
A. J. Thomson,
B. C. Berks, and D. J. Richardson.
1998.
Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport.
J. Biol. Chem.
273:28785-28790[Abstract/Free Full Text].
|
| 21.
|
Saraste, M., and J. Castresana.
1994.
Cytochrome oxidase evolved by tinkering with denitrification enzymes.
FEBS Lett.
341:1-4[CrossRef][Medline].
|
| 22.
|
Schulz, H.,
R. A. Fabianek,
E. C. Pellicioli,
H. Hennecke, and L. Thöny-Meyer.
1999.
Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB.
Proc. Natl. Acad. Sci. USA
96:6462-6467[Abstract/Free Full Text].
|
| 23.
|
Shapleigh, J. P., and W. J. Payne.
1985.
Nitric oxide-dependent proton translocation in various denitrifiers.
J. Bacteriol.
163:837-840[Abstract/Free Full Text].
|
| 24.
|
Simon, R.,
V. Priefer, and A. Pühler.
1983.
Vector plasmids for in vivo and in vitro manipulations of gram-negative bacteria, p. 99-108.
In
A. Pühler (ed.), Molecular biology of bacteria and plant interaction. Springer-Verlag KG, Berlin, Germany.
|
| 25.
|
Stoll, R.,
M. D. Page,
Y. Sambongi, and S. J. Ferguson.
1996.
Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.
Microbiology
142:2577-2585[Abstract].
|
| 26.
|
van der Oost, J.,
A. P. de Boer,
J. W. de Gier,
W. G. Zumft,
A. H. Stouthamer, and R. J. van Spanning.
1994.
The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.
FEMS Microbiol. Lett.
121:1-9[Medline].
|
| 27.
|
van Spanning, R. J.,
E. Houben,
W. N. Reijnders,
S. Spiro,
H. V. Westerhoff, and N. Saunders.
1999.
Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans.
J. Bacteriol.
181:4129-4132[Abstract/Free Full Text].
|
| 28.
|
van Spanning, R. J.,
C. W. Wansell,
W. N. Reijnders,
N. Harms,
J. Ras,
L. F. Oltmann, and A. H. Stouthamer.
1991.
A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.
J. Bacteriol.
173:6962-6970[Abstract/Free Full Text].
|
| 29.
|
Watmough, N. J.,
G. Butland,
M. R. Cheesman,
J. W. Moir,
D. J. Richardson, and S. Spiro.
1999.
Nitric oxide in bacteria: synthesis and consumption.
Biochim. Biophys. Acta
1411:456-474[Medline].
|
| 30.
|
Watmough, N. J.,
M. R. Cheesman,
C. S. Butler,
R. H. Little,
C. Greenwood, and A. J. Thomson.
1998.
The dinuclear center of cytochrome bo3 from Escherichia coli.
J. Bioenerg. Biomembr.
30:55-62[CrossRef][Medline].
|
| 31.
|
Watmough, N. J.,
M. R. Cheesman,
R. B. Gennis,
C. Greenwood, and A. J. Thomson.
1993.
Distinct forms of the haem o-Cu binuclear site of oxidised cytochrome bo from Escherichia coli. Evidence from optical and EPR spectroscopy.
FEBS Lett.
319:151-154[CrossRef][Medline].
|
| 32.
|
Zaslavsky, D., and R. B. Gennis.
1998.
Substitution of lysine-362 in a putative proton-conducting channel in the cytochrome c oxidase from Rhodobacter sphaeroides blocks turnover with O2 but not with H2O2.
Biochemistry
37:3062-3067[CrossRef][Medline].
|
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Abstract
Introduction
Present address: Department of Biochemistry, University of Oxford,
Oxford OX1 3QU, United Kingdom.
