Characterization of the Shewanella oneidensis MR-1 Decaheme Cytochrome MtrA
EXPRESSION IN ESCHERICHIA COLI CONFERS THE ABILITY TO REDUCE SOLUBLE FE(III) CHELATES*
Katy E. Pitts
,
Paul S. Dobbin
,
Francisca Reyes-Ramirez
,
Andrew J. Thomson ¶,
David J. Richardson
and
Harriet E. Seward
¶ ||
From the
Centre for Metalloprotein Spectroscopy and Biology,
School of Biological Sciences and
¶ School of Chemical Sciences, University of East
Anglia, Norwich NR4 7TJ, United Kingdom and
Department of Biological Sciences, University of
Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom
Received for publication, March 13, 2003
, and in revised form, May 1, 2003.
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ABSTRACT
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Shewanella oneidensis MR-1 has the metabolic capacity to grow
anaerobically using Fe(III) as a terminal electron acceptor. Growth under
these conditions results in the de novo synthesis of a number of
periplasmic c-type cytochromes, many of which are multiheme in nature
and are thought to be involved in the Fe(III) respiratory process. To begin a
biochemical study of these complex cytochromes, the mtrA gene that
encodes an approximate 32-kDa periplasmic decaheme cytochrome has been
heterologously expressed in Escherichia coli. Co-expression of
mtrA with a plasmid that contains cytochrome c maturation
genes leads to the assembly of a correctly targeted holoprotein, which
covalently binds ten hemes. The recombinant MtrA protein has been
characterized by magnetic circular dichroism, which shows that all ten hemes
have bis-histidine axial ligation. EPR spectroscopy detected only
eight of these hemes, all of which are low spin and provides evidence for a
spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA
have been carried out with optical- and EPR-monitored methods, and the hemes
are shown to reduce over the potential range 100 to 400 mV. In
intact cells of E. coli, MtrA is shown to obtain electrons from the
host electron transport chain and pass these onto host oxidoreductases or a
range of soluble Fe(III) species. This demonstrates the promiscuous nature of
this decaheme cytochrome and its potential to serve as a soluble Fe(III)
reductase in intact cells.
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INTRODUCTION
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Bacterial iron respiration is the process in which the Fe(III) acts as the
terminal electron acceptor in an energy-conserving bacterial respiratory
electron transport chain. It is an anaerobic process that provides a bacterium
with a means of respiration when oxygen is absent and is distinct from the
process of iron assimilation in which iron is taken up into the cell by
energy-consuming systems and incorporated in to cell biomass. The capacity for
Fe(III) respiration is phylogenetically widespread, and the ecological impact
in microoxic and anoxic soils and sediments is considerable
(16).
A particular problem for Gram-negative bacteria is that at circum-neutral pH,
the speciation of Fe(III) is complex because the cation is present as either
insoluble polynuclear oxo/hydroxo-bridged complexes or as soluble organic
chelates. Thus, depending on the environment, Fe(III) is presented to bacteria
in a range of forms that can vary in solubility, steric properties, and
overall charge. The best characterized group of Fe(III)-respiring bacteria are
species of the genus Shewanella, which are widespread facultative
anaerobes of the
-proteobacteria group that can express a number of
terminal respiratory reductases including nitrate, nitrite, fumarate, and
trimethylamine N-oxide reductases
(7). During growth under
iron-respiring conditions, de novo synthesis of a number of multiheme
c-type cytochromes that may a play a role in the Fe(III) respiration
process occurs. Cytochrome oxidation has been observed during Fe(III)
reduction, and a number of genetic loci that encode multiheme cytochromes have
been implicated as being important for Fe(III) respiration
(811).
These include cymA, which encodes an inner membrane periplasmic
tetraheme quinol dehydrogenase (CymA), and the mtrDEF-OmcA-MtrCAB
gene cluster, which encodes three outer membrane decaheme cytochromes (MtrF,
OmcA, and MtrC), two periplasmic decaheme cytochromes (MtrD and MtrA), and two
putative outer membrane
-barrel proteins (MtrE and MtrB). In addition
some small periplasmic tetraheme cytochromes such as the small tetraheme
cytochrome c and iron-induced flavocytochrome c3
may also play a role in Fe(III) respiration
(12,
13). Consideration of the
different cellular locations of these cytochromes has led to the proposal of a
mechanism of electron transfer for iron respiration in which electrons are
transferred from quinol via CymA to periplasmic multiheme cytochromes
(10,
11,
14). These periplasmic
cytochromes may then be able to nonspecifically reduce soluble Fe(III)
chelates, but in order to reduce insoluble Fe(III) complexes, the electrons
must then be passed to outer membrane cytochromes, e.g. OmcA.
Much of the work that has led to the development of this model for Fe(III)
respiration has been done on two species, Shewanella frigidimarina
NCMIB400 and Shewanella oneidensis MR-1. The genome sequence of the
latter has recently been completed and reveals that there are around forty
genes encoding c-type cytochromes in this organism, many of which are
predicted to be multiheme in character
(15). The large number of
c-type cytochromes synthesized during growth in the presence of
Fe(III) makes it difficult to assess the properties and function of individual
cytochromes in vivo. In this work, we have thus begun to resolve the
properties and functions of individual cytochromes in Fe(III) respiration
through heterologous expression of mtrA in Escherichia coli
with the aim of providing both large quantities of protein for biophysical
analysis and insight into any new metabolic capacity that the expression of
the gene confers.
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EXPERIMENTAL PROCEDURES
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Expression of mtrA in E. coliThe mtrA gene (GenBankTM
accession number AF083240
[GenBank]
) was amplified by PCR using genomic DNA prepared
from S. oneidensis MR-1 as the template, suitable homologous primers,
and TaqDNA polymerase. An AccepTor VectorTM kit (Novagen and CN
Biosciences) was used to ligate the 1-kb PCR product into the
-galactosidase gene of pETBlue-1, now called pKP1.
Plasmid DNA was prepared from the resulting white colonies and checked by
single and double digestions with PstI and NcoI, which both
cut one restriction site each on the mtrA gene. Expression host
cells, E. coli JM109(DE3), were transformed first with the vector
pEC86 containing the ccmABCDEFGH (cytochrome c
maturation) genes (16). These
cells were grown and subsequently transformed with pKP1.
Growth ConditionsCells of the E. coli JM109(DE3)
(pKP1+pEC86) strain were grown aerobically at 37 °C in
Luria-Bertani medium (10 g/liter tryptone, 10 g/liter NaCl, 5 g/liter of yeast
extract, pH adjusted with NaOH to 7.5) until they reached an optical density
of approximately 1.0. They were then induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside and grown under the
same conditions overnight.
Cell Fractionation and Protein PurificationCells were
harvested at 7000 x g and 4 °C for 10 min and resuspended
in 100 mM NaHepes at pH 7.5 (0.3 g of wet cells
ml1). The cells were treated with polymixin B (1
mg ml1) to break the outer membrane and were
incubated at 37 °C for 1 h. The lysed cells were spun at 15,000 x
g and 4 °C for 45 min. The resulting periplasmic fraction was
found to contain the c-type cytochrome and was loaded onto a
DEAE-Sepharose (fast flow) anion exchange column, which had been equilibrated
with 20 mM Tris-HCl, pH 8.0. After washing with 2 column volumes of
buffer, MtrA was eluted under a linear gradient of 0250 mM
over 4 column volumes. Fractions were combined and dialyzed against 20 mm
Tris-HCl, 50 mM NaCl, pH 8.0, overnight. MtrA was further purified
by fast protein liquid chromatography at room temperature on a HiLoad 16/60
Superdex 75-gel filtration column. Prior to spectroscopic characterization,
MtrA was concentrated and buffer exchanged into 20 mM Hepes buffer,
pH 7.0, by ultrafiltration with a 10-kDa cut-off membrane.
General Analytical Procedures and Cytochrome Oxidation
Kinetics Analyses of the cell extracts and purified proteins were
routinely performed with SDS-PAGE. Gels were examined for the presence of
c-type cytochromes by heme-linked peroxidase staining as described
previously (17). For
quantification of c-type hemes, 0.5 ml of a stock solution containing
200 mM NaOH and 40% by volume pyridine was placed in a cuvette with
0.5 ml of cytochrome solution, 3 µl of 0.1 M ferricyanide were
added, and the spectrum was recorded. Sodium dithionite was then added to
reduce the sample. The extinction coefficient of the reduced minus oxidized
sample at 550535 nm was taken as 24 mm1
cm1
(18). Oxidation kinetics of
reduced MtrA following addition of different electron acceptors was followed
at 552 nm. Iron chelates were prepared as described by Dobbin et al.
(19).
UV-visible, EPR, and Magnetic Circular Dichroism
Spectroscopies Absorption spectra of whole cells or purified MtrA
were obtained on an SLM Aminco DW-2000 or a Hitachi U4001 UV-visible
spectrometer. EPR spectra were obtained on a Bruker EMX system X-band
spectrometer equipped with an Oxford Instrument ESR-9 liquid helium flow
cryostat and a dual mode EPR cavity. Spin quantification of the EPR spectra
was carried out by integration the gz peaks in comparison with a
Cu(II)(EDTA) standard (1 mM) using the method of Aasa and
Vanngård (20). Where
there was overlap of the gz features, the peaks were first
deconvoluted with gaussian fits using WinEPR software from Bruker. Magnetic
circular dichroism spectra were measured with circular dichroism spectrometers
from Jasco with models J800 and J730 being employed for the wavelength ranges
of 240800 and 7002000 nm, respectively. The magnetic field was
applied with a SM-1 super-conducting solenoid with a 25-mm room temperature
bore from Oxford Instruments, which generates a maximum field of 6 Tesla.
Optical Redox TitrationMediated spectrophotometric redox
potentiometry was undertaken using methodology described by Dobbin et
al. (13) Titrations with
dithionite on 0.2 µM MtrA protein were performed under argon
atmosphere at 15 °C in 20 mM NaHepes, pH 7.5. Mediators
(duroquinone, phenazine ethosulfate, phenazine methosulfate,
anthraquinone-2-sulfonic acid, antraquinone-2,6-disulfonic acid, menadione,
and benzyl viologen) were used at a concentration of 10 µM.
Reduction potentials are referenced to standard hydrogen electrode.
Potentiometric Titration Monitored by EPRUnder a nitrogen
atmosphere, mediated redox potentiometry was performed using a polished
graphite electrode in the form of a carbon pot in the range of 50273
mV. Samples were allowed to equilibrate in the carbon pot for 12 h,
removed in EPR tubes under nitrogen, and frozen immediately. Potential drift
was monitored and was <5 mV in all of the cases. Mediators, which were used
at a concentration of 10 or 30 µM, were duroquinone, menadione,
anthraquinone-2-sulfonic acid, safranine O, methyl, and benzyl viologen. In
the range below 273 mV, dithionite was used as a reductant in a similar
manner to the optical redox titration because of the difficulty of poising the
samples with the carbon electrode alone.
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RESULTS AND DISCUSSION
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De novo synthesis of Periplasmic c-Type Cytochromes during Growth of S.
oneidensis on Soluble Fe(III) Species and Heterologous Expression of mtrA in
E. coliThe c-type cytochrome profile of periplasmic
fractions prepared from S. oneidensis MR-1 was compared in cells
grown microaerobically or anaerobically with a variety of respiratory electron
acceptors. Hemestained SDS-PAGE gels revealed that a large number of different
periplasmic cytochromes were synthesized in the cells grown with Fe(III) by
comparison with the other growth conditions
(Fig. 1A). Of
particular note is the de novo synthesis of
35-kDa
cytochrome(s). The genome of S. oneidensis MR-1 encode four putative
paralogues of a decaheme 35-kDa c-type cytochrome including two, MtrA
and MtrD, that are part of the mtrDEF-omcA-mtrCAB gene cluster and
that are known to be important in Fe(III) respiration
(11). No detailed biochemical
characterization of any of these periplasmic decaheme cytochromes has been
reported to date, and thus to investigate the biophysical and functional
properties of this family of cytochromes, the gene for mtrA was
selected for heterologous expression in E. coli.

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FIG. 1. SDS-PAGE analysis of S. oneidensis MR-1 and E. coli
JM109(DE3) cell extracts and purified MtrA stained for heme-dependent
peroxidase activity. A, 10% SDS-PAGE gel loaded with periplasmic
extracts from S. oneidensis grown microaerobically (lane 3)
or anaerobically with fumarate (lane 2) or Fe(III) citrate (lane
1) present as electron acceptor. B, 15% SDS-PAGE gel loaded with
periplasmic fractions from E. coli JM109(DE3) (lane 1),
JM109(DE3) (pEC86) (lane 2), JM109(DE3) (pEC86 +
pKP1)(lane 3) and membrane fractions from E. coli
JM109(DE3) (lane 4), JM109(DE3) (pEC86) (lane 5),
and JM109(DE3) (pEC86 + pKP1)(lane 6). B,
visible absorption spectrum of intact cells ( 0.5 mg dry weight
ml1) of JM109(DE3) (bottom trace),
JM109(DE3) (pEC86)(middle trace), and JM109(DE3)
(pEC86 + pKP1) (top trace).
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The mtrA gene was amplified from chromosomal DNA prepared from
S. oneidensis MR-1 and cloned into pETBlue-1 to yield pKP1.
This and pEC86, which contains genes that encode cytochrome
c maturation
(Ccm)1 proteins and
that are required for the covalent attachment of heme via thioether linkages
to polypeptide chains in the periplasmic compartment, was transformed into
E. coli JM109(DE3). Colonies of these cells were notably pink in
color. Visible spectra of aerobically grown cultures of E. coli
JM109(DE3) (pKP1+pEC86) collected between 500 and 600 nm
revealed intense absorption bands at
550 nm
(Fig. 1C). This is
characteristic of the
-absorbance band of reduced c-type
cytochrome and was absent from E. coli JM109(DE3), E. coli
JM109(DE3) (pEC86), and E. coli JM109(DE3) (pKP1),
all of which exhibited weak absorption bands at 560 nm that are characteristic
of reduced b-type cytochromes and are likely to arise from endogenous
cytochromes such as the cytochrome bo oxidase. Periplasmic and
membrane fractions were prepared from aerobic cultures of E. coli
JM109(DE3) (pKP1+pEC86) that had been induced with
isopropyl-1-thio-
-D-galactopyranoside. Heme-stained SDS-PAGE
gels of these fractions revealed the presence in both fractions of a 35-kDa
heme-staining band that is the expected size of MtrA
(Fig. 1B). This band
was absent in E. coli JM109(DE3), E. coli JM109(DE3)
(pKP1) (data not shown), and E. coli JM109(DE3)
(pEC86) (Fig
1B). A 17-kDa heme-staining band was also present in the
membrane fractions of JM109(DE3) (pEC86) and JM109(DE3)
(pKP1 + pEC86) but absent from JM109(DE3). This corresponds
to the size of the heme-binding cytochrome c maturation protein CcmE,
which is membrane-associated and encoded by pEC86. Additional faint
bands at 24 kDa could be seen in membranes of JM109(DE3) (pEC86) and
JM109(DE3) (pKP1 + pEC86), which correspond to the size
expected of the membrane-anchored tetraheme quinol dehydrogenase, NapC, of the
NaAB nitrate reductase system. A heme-staining band at around 55 kDa was
present in all of the periplasm and membrane samples. This corresponds to the
periplasmic pentaheme NrfA nitrite reductase protein. The observation that
some of the periplasmic NrfA "sticks" to membrane preparations has
been reported previously (17).
The nap and nrf genes are characteristically expressed
during anaerobic growth of E. coli. The synthesis of the Nap and Nrf
cytochromes revealed by heme staining was confirmed by direct enzymological
assays for nitrate and nitrite reductase and suggests that a low level of
nap and nrf expression is occurring as a result of the
cultures becoming oxygen-limited at high cell densities.
Taken together, the data from the heme-stained SDS-PAGE analysis suggest
that E. coli can express mtrA and correctly locate and
assemble holo-MtrA in the periplasm when mtrA is co-expressed with
the ccm genes in E. coli JM109(DE3) (pKP1 +
pEC86). Similar to NrfA, it seems that some MtrA will also stick to
membranes and this finding suggests that it can associate with the periplasmic
face of either or both inner and outer membranes. Quantification of the
reduced absorption band at 552 nm in the intact cells that have synthesized
MtrA with an extinction coefficient (derived from purified protein) of 280
mM1 cm1
(28 mM1 cm1/heme)
reveals that around 6 nmol protein (mg cells)1 is
produced.
Purification and Characterization of MtrAThe recombinant
35-kDa MtrA was purified using a combination of anion exchange and
gel-filtration chromatographies (see "Experimental Procedures").
The protein stained poorly with Coomassie Blue but was judged to be pure by
the absence of additional bands on Coomassie Blue-stained gels
(Fig. 2A,
inset). Purified protein has a ratio of
A408/A280 nm of
4.5. An analysis
of the native protein in solution by dynamic light scattering revealed the
MtrA samples to be monodisperse with an approximate molecular mass of 30 kDa,
suggesting that it is monomeric in solution. An analysis using the program
SignalP (21) suggested a
cleavage site for the signal peptide sequence after position 34 in the MtrA
pre-protein amino acid sequence (Fig.
9). This would suggest a processed molecular mass of 32,422 Da for
the apopeptide and a total mass of 38,584 Da with the 10 predicted c
hemes. An analysis of MtrA by MALDI-TOF mass spectrometry gave a molecular
mass value of 38,581 kDa, and this suggests that the heterologously
synthesized protein does indeed bind ten hemes.

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FIG. 2. UV-visible spectropotentiometric analysis of MtrA (reductive and
oxidative titration). A, spectrum of oxidized (solid
line) and fully reduced (dotted line) purified MtrA protein,
concentration of 15 µM by protein, in 50 mM Hepes
buffer, pH 7.0. B, plot of absorption at 552 nm as a function of
potential (corrected for dilution). The solid line represents a fit
to two n = 1 Nernstian components with
Em values of 200 and 375 mV (70:30%
total absorbance change). Inset, SDS-PAGE gel with prestained
Coomassie Blue markers of 250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa,
heme-stained purified recombinant MtrA, and Coomassie Blue-stained recombinant
MtrA.
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FIG. 9. Sequence alignment of MtrA from S. oneidensis and E.
coli NrfB (35,
36). The CXXCH
heme binding motifs are numbered, and conserved His residues in MtrA
are denoted by +.
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Optical Redox TitrationThe optical spectrum of oxidized
MtrA has a Soret (
) band centered at 408 nm, a visible region peak at
531 nm and a shoulder at 560 nm. The spectrum is typical of low spin ferric
heme. Upon reduction, the Soret band
max shifts to 420 nm
and
- and
-peaks at 525 and 552 nm that are characteristic of low
spin ferrous c-type cytochromes appear
(Fig. 2A). Pyridine
hemochromogen assays give a value of
132
mM1
cm1/heme for the MtrA Soret band, which is a
value typical of low spin hemoproteins
(18,22).
This would correspond to an extinction coefficient of 1,320
mM1 cm1
for the entire protein. Absorption spectra of MtrA were collected at a range
of potentials between +400 and 450 mV. An increase in absorbance at 552
nm was observed over the range of approximately 100 to 450 mV.
The change in absorption at 552 nm is best fitted to two groups of hemes that
titrate as n = 1 components and that account for 30 and 70% of the
total absorption change with midpoint potentials,
Em, of 375 and 200 mV
(Fig. 2B). This simple
fit suggests that three hemes have near iso-midpoint potentials at 375
mV and seven hemes have near iso-midpoint potentials at 200 mV. These
values are in the range that is commonly found for bis-His ligated
hemes as a result of the electron-donating properties of the imidazole ring
nitrogens, which serve to stabilize the oxidized state
(22).
Magnetic Circular Dichroism of MtrAThe size and position of
signals in UV-visible region of the spectrum, which is shown in
Fig. 3A, is consistent
with ferric low spin heme, with a Soret derivative cross-over at 407 nm and a
peak-to-trough intensity of 97 mm1
cm1/heme. In fact, the Soret and visible region
is very similar to other bis-histidine-ligated multiheme cytochromes
such as CymA and OmcA from S. frigidimarina NCIMB400
(23). The charge-transfer band
for low spin ferric heme occurs in the NIR region (8002500 nm), and the
peak wavelength is an indicator of the axial ligands to the heme iron
(24,
25). The NIR spectrum of MtrA
shows a positive LMCT (CTls) band at 1490 nm with vibrational side
structures to higher energy (Fig.
3B). The position of the CTls maximum at 1490
nm is consistent with low spin ferric heme with bis-histidine
ligation. Histidine/lysine or histidine/amine coordination could also give
rise to a CTls band of similar position and intensity, but the EPR
of amine-bound hemes is quite unique in shape and position
(24) and the EPR of MtrA is
not consistent with lysine ligation (see below). The intensity of this
CTls band is also consistent with an almost 100% population of low
spin heme.

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FIG. 3. Room temperature (RT) magnetic circular dichroism
(MCD) of MtrA in 50 mM Hepes buffer, pH* 7.2, in
D2O. A, Soret and visible region. B,
near infrared region. Intensities are given per heme. Concentration of protein
by heme: Soret region, 115 µM; visible region, 350
µM; NIR region 1.6 mM.
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Electron Paramagnetic Resonance of Oxidized MtrAThe 10 K
perpendicular mode X-Band EPR spectrum of air-oxidized MtrA
(Fig. 4A) contains a
number of low spin (S = 1/2) rhombic species. Normally three features
are observed in rhombic spectra, but in these spectra, only the
gmax (gz) features are clearly observed. The second
feature is an overlay of at least two gy features, and the third
feature is also obscured, either because of its width or by other features.
The two major species have gmax values of 2.95 and 2.88 and a
second feature in the derivative at g
2.27. The third feature is
broadened and is not observed. These g-values are consistent with
bis-histidine ligation at the heme in which the histidine imidazole
ring planes are approximately parallel to each other. Spin integration of the
signals reveals that these two major low spin species each account for
40% of the heme population. There is also a positive feature at g = 2.43
that is likely to arise from a small population of low spin
histidine/hydroxide heme, probably from a heme or hemes in which one of the
histidine ligands has become detached, and a derivative feature at g = 2.06,
which may be assigned to adventitious copper.

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FIG. 4. X-band perpendicular mode EPR of MtrA. Spectra were collected at 10
K. Microwave frequency, 9.66 GHz; microwave power, 2 milliwatts; modulation
amplitude, 10 G. A, air-oxidized spectrum. B, redox
titration of MtrA. Solid line 65 µM, 53 mV;
dotted line, 43 µM, 118 mV; dashed
line, 43 µM, 183 mV; dot dash, 65
µM, 273 mV; dotdot dash, 65 µM,
298 mV; solid line, 32 g(m)M, 453 mV, short dotted
line, 39 µM. Intensities are corrected for changes in gain,
concentration, and sample volume. C, fit of EPR titration points
(x = 1). Solid line, g = 2.95; dotted line,
g = 1.80. Nernstian fits of two n = 1 components gave
Em values of 136 and 288 mV for the
g = 2.95 peak (70:30% total change in peak height). The majority of the g =
1.80 trough titrates away with an Em of 88
mV
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Approximately 20% of the heme population is unaccounted for by the
integrations of the low spin features. This population of heme may account for
a pair of spin-coupled hemes, which could be EPR silent. However and notably,
there is a broad trough at g = 1.80 that could in principle arise from a
weakly coupled heme pair of low spin paramagnets. Parallel mode EPR spectra of
MtrA were run to examine whether any integer spin systems could be detected,
but no signals were observed. Examples of S = 1/2 spin-coupled
systems have been detected in the multiheme hydroxylamine-cytochrome
c oxidoreductase and have similar spectral features
(26).
EPR-monitored Potentiometric Titration of MtrAA
potentiometric titration of MtrA monitored by EPR was carried out to probe for
additional heme species or extra intensity that could occur as magnetically
interacting species are reduced (Fig.
4B). The addition of mediators (detailed under
"Experimental Procedures") did not change the EPR spectrum of the
oxidized protein. However, at 53 mV, a potential at which the optical
redox titer does not show any reduction of the heme, the gmax at
2.88 has diminished, whereas the gmax at 2.95 has increased and now
accounts for 80% of the heme concentration. This may indicate a
redox-dependent conformational change in the protein that has a knock-on
effect on the heme ligand geometry. At the same potential, the broad trough at
g = 1.80 has decreased in intensity and the adventitious copper signal at g =
2.06 in the oxidized spectrum has been reduced out of the spectrum. With
further reduction, the low spin rhombic trio at g = 2.95 and g = 2.28 continue
to diminish (the third feature expected at approximately g = 1.5 is not
observed). Thus at 118 mV, the 2.95 peak integrates to 50% of the heme
population but accounts for only 5% of the heme content at 298 mV. No
new species are observed in the poised samples. At 453 mV, there is a
radical species at g = 2.0 in the spectrum that is likely to arise from
reduced methyl or benzyl viologen.
The titer of the g = 2.95 signal and the broad trough at g = 1.80 are shown
in Fig. 4C. Note that
as the EPR titer measures the decrease in peak height, the y axis
points have been multiplied by 1. The EPR titers follow a similar trend
to the optical titer, but both sets of data have more positive midpoint
potentials, particularly the g = 1.80 trough in which 80% of its intensity is
lost with a Em of 82 mV. The peak at g =
2.96 fits to two sets of heme with midpoint potentials of 136 and
288 mV (Fig.
4C). These Em values, which
are offset relative to the optical titer, could be the result of collecting
the spectra at 10 K. However, they are in the same potential domain as the
data collected for the visible spectropotentiometic titrations and are still
entirely consistent with the presence of low spin bis-His ligated
hemes.
Catalytic Properties of MtrAThe catalytic properties of
reduced MtrA were assessed by monitoring the oxidation kinetics of the hemes
in the presence of different electron acceptors. As expected from the low
redox potentials of the hemes, reduced MtrA was rapidly oxidized by air.
Consequently, all of the experiments were carried out under strict anaerobic
conditions in a glove box. Oxidation of the reduced MtrA was monitored at 552
nm, and the experiments revealed that all of the ferrous hemes were rapidly
oxidized upon addition of Fe(III) NTA, Fe(III) EDTA, or Fe(III) Maltol
(Fig. 5A, shown for
Fe(III) EDTA only). The addition of fumarate resulted in no re-oxidation,
whereas the addition of nitrite and nitrate resulted in a slow re-oxidation
that was 12 orders of magnitude slower than that of Fe(III) EDTA for
which a rate of approximately 5 nmol of electrons/s was obtained
(Fig. 5A).

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FIG. 5. Oxidation kinetics of reduced MtrA following addition of different
electron acceptors. A, prereduced MtrA (20 µm) was incubated
anaerobically, and NaNO3, NaNO2, or Fe(III) Maltol was
added to a final equimolar concentration of 0.2 mM at the time
indicated by the arrow. B, top trace, MtrA was preincubated with 0.2
mM NaNO3 and periplasmic nitrate reductase (NapAB) was
added (100 nm) at the time indicated by the arrow. B, bottom trace,
MtrA was preincubated with 0.2 mM NaNO2, and periplasmic
nitrite reductase (NrfA) was added (100 nm) at the time indicated by the
arrow.
|
|
Electron Transport to Fe(III) in Intact Cells of E.
coliMtrA could play two roles in Fe(III) respiration by S.
oneidensis MR-1: (i) participation in intermembrane electron transfer
between the inner membrane quinol dehydrogenase (CymA) and the outer membrane
cytochromes for reduction of insoluble Fe(III) species and (ii) direct
reduction of soluble Fe(III) chelates using electrons derived from inner
membrane quinols via the tetraheme quinol dehydrogenase CymA
(Fig. 6A,
scheme). E. coli has a homologue of CymA, NapC, which is
involved in menaquinol oxidation as part of the NapAB nitrate reductase system
(27,
28). Thus the possibility that
the "foreign" periplasmic MtrA could mediate electron transport
from the respiratory components of the E. coli inner membrane to
soluble Fe(III) species in intact cells was investigated. Cells of E.
coli JM109(DE3) (pKP1 + pEC86) were incubated under
anaerobic conditions with lactate as electron donor. Spectral analysis
confirmed that MtrA was reduced under these conditions as judged by the
reduced absorbance maximum at 552 nm (Fig.
7A). This confirmed that E. coli could transport
electrons from physiological electron donors to the S. oneidensis
MtrA. Following the addition of Fe(III) NTA, rapid oxidation of the MtrA hemes
was observed. Inspection of the spectrum after oxidation revealed that only
the absorption peak at 552 nm disappeared
(Fig. 7). The reduced
cytochrome peaks at 560 nm that arise from the endogenous b-type
cytochromes remained, and control experiments in which Fe(III) NTA was added
to E. coli JM109(DE3) confirmed that the chelate did not oxidize the
endogenous cytochromes (Fig.
7B). Following the consumption of the Fe(III) NTA, full
re-reduction of the MtrA cytochromes in E. coli JM109(DE3)
(pKP1 + pEC86) occurred. This took place without the need
for addition of exogenous artificial electron donor and suggests that
re-reduction was occurring via electron transport through the host respiratory
system. The kinetics of the cytochrome oxidation and re-reduction was
monitored as a function of time at 552 nm. The duration of the cytochrome
oxidation period was proportional to the amount of Fe(III)NTA added, and this
corresponds to a steady state rate of Fe(III) reduction of
10 nmol (mg
dry weight cells)1
min1, which compares to around 90 nmol (mg dry
weight cells)1 min1
for Fe(III) NTA in S. frigidimarina
(13,
19). When the
semi-napthoquinone analogue
2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was added to
the anaerobic cells prior to the addition of Fe(III) NTA, rapid oxidation of
the MtrA hemes still occurred but re-reduction was inhibited
(Fig. 8). Thus the duration of
cytochrome oxidation for equimolar pulses of Fe(III) NTA was approximately six
times longer in the presence of HQNO. The same result was observed when HQNO
was added to cells of S. frigidimarina NCIMB400 that are respiring
Fe(III) (13). These results
suggest that E. coli can feed electrons from the quinol pool to MtrA,
which can then mediate electron transfer to soluble Fe(III) species. An
analysis of E. coli membranes established that the 25-kDa CymA
homologue, NapC, was present in the JM109(DE3) (pKP1 +
pEC86) cells used in these experiments; therefore, given the
sensitivity to HQNO, this is the most probable route of electron transfer to
MtrA. Other Fe(III) species that could oxidize the MtrA hemes when added to
intact cell suspensions of E. coli JM109(DE3) (pKP1 +
pEC86) included Fe(III) EDTA, Fe(III) Maltol, and Fe(III)
benzohydroxamic acid. However, Fe(III) citrate was not a good electron
acceptor. This has also been previously observed with R. capsulatus,
which like E. coli JM109(DE3) (pKP1 + pEC86) can
reduce a range of soluble Fe(III) species
(29). This may be because at
the high concentrations added, Fe(III) citrate forms complex polynuclear
species that cannot access the periplasm. Insoluble iron oxides were also
unable to oxidize the MtrA in intact cells.

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FIG. 6. A possible scheme for electron transfer in S. oneidensis MR-1
and E. coli mediated by S. oneidensis MtrA. Upper
panel, S. oneidensis MR-1. Lower panel, E. coli. The
arrows indicate possible routes of electron flow. Black
circles indicate heme groups.
|
|

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FIG. 7. Visible absorption spectra of anaerobic intact cell suspensions of
E. coli. A, E. coli JM109(DE3) (pKP1 +
pEC86). Sold line, anaerobic suspension; dashed
line, anaerobic suspension 2 min after the addition of Fe(III) NTA (0.1
mm). B, E. coli JM109(DE3). Sold line, anaerobic suspension;
dashed line, anaerobic suspension 2 min after the addition of Fe(III)
NTA (0.1 mm). Cells were suspended in a lactate (50 mM), NaHepes
(100 mM), pH 7.0, medium to a concentration of 0.3 mg dry
weight ml1.
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FIG. 8. The kinetics of MtrA oxidation and re-reduction measured at 552 nm in
anaerobic intact cell suspensions of E. coli JM109(DE3)
(pKP1 + pEC86). A, MtrA oxidation and
reduction following addition of Fe(III) NTA (0.1 mM). B,
MtrA oxidation and reduction following addition of Fe(III) NTA (0.1
mM) to cells preincubated for 10 min with HQNO (50 µm). Cells
were suspended in a lactate (50 mM), NaHepes (100 mM),
pH 7.0, medium to a concentration of 0.3 mg dry weight
ml1
|
|
MtrA oxidation in intact cells was also observed after the addition of
either nitrate or nitrite to anaerobic suspensions (data not shown). The rates
were comparable to those for Fe(III) reduction. This contrasted to the
observations made with purified MtrA, which showed that the rate of MtrA
oxidation by nitrate and nitrite was much slower than for the oxidation by
Fe(III) species (as already shown in Fig
5A). To study this further, reconstitution experiments of
purified MtrA with purified NapAB nitrate reductase or NrfA nitrite reductase
showed that it could serve as a poor electron donor to periplasmic nitrate
reductase but a good electron donor to NrfA
(Fig. 5B). As detailed
earlier, enzymatic and heme-stained SDS-PAGE analysis
(Fig. 1) revealed that the
periplasmic nitrate and nitrite reductase systems were both present in the
cells used in these experiments, and thus the rapid rate of oxidation of MtrA
by nitrate and nitrite in intact cells can be explained as a result of these
systems drawing electrons from MtrA during the reduction of nitrate to
ammonium (Fig. 8).
This demonstrates that in the intact E. coli cells the foreign
MtrA can integrate into the endogenous electron transport system of the
host.
 |
CONCLUSIONS
|
---|
This work has reported the first detailed biochemical characterization of a
member of the MtrA family of periplasmic decaheme cytochromes. The
characterization has been facilitated through the successful heterologous
expression of mtrA in E. coli, which when co-expressed with
ccm genes results in the assembly of correctly targeted periplasmic
decaheme cytochrome. Spectroscopic characterization has suggested that all of
the heme iron ions in ferric MtrA are low spin hexacoordinate species in which
histidine ligands provide both the distal and proximal Fe(III) ligands. The
proximal ligands will be provided by each of the 10 CXXCH heme
binding motifs, but an additional ten His ligands are then required for each
distal site. Analysis of the S. oneidensis genome sequence reveals
four paralogs of MtrA (15),
the gene products of mtrD, so1427, and so4360. Alignment of
the four primary structures reveals that all ten of the CXXCH heme
binding motifs align and that an additional 11 conserved His residues can be
identified (Fig. 9). If heme
binding sites 34, 56, and 78 are considered separately,
two conserved His are found to be in an arrangement of
CXXCHX1012HX79CXXCHXXH.
This arrangement can also be identified between hemes 2 and 3 in the small
tetraheme cytochromes and the tetraheme domains of the flavocytochrome
c fumarate reductases of S. frigidimarina NCIMB400 and
S. oneidensis MR-1. Structures determined for these proteins
(12,
3032,
34) reveal that the two hemes
bound by this motif form a near parallel heme pair in which the heme irons are
around 4-Å apart, and the conserved His between the CXXCH
motifs serves as a distal ligand for the heme iron bound by the first motif
and the conserved His after the second heme binding motif serves as a distal
ligand for another heme iron in the multiheme protein
(32,
34). Thus, the present
sequence analysis of the MtrA paralogues strongly suggests that a number of
the 10 hemes could be organized into these parallel heme pair arrangements.
The first
190 amino acids of the decaheme MtrA paralogues contains five
CXXCH heme binding motifs. If these are aligned with the
190
amino acids of the pentaheme NrfB protein, with which MtrA shares a low
sequence identity (35) and
which is thought to serve as an electron donor to the NrfA nitrite reductase
of E. coli (36), it
can be seen that the spatial distribution of the CXXCH motifs in the
primary structure are very similar, including the
CXXCHX1012HX79CXXCHXXH
arrangement between hemes 3 and 4. This suggests that these polypeptides will
fold in a similar manner. The crystal structure of the NrfA nitrite reductase
(33) shows that the protein is
an
110-kDa decaheme homodimer and has raised the possibility that the
electron donating NrfB also binds to this complex as a decaheme homodimer.
This dimer would have a molecular mass of around 35 kDa, similar in size to
the decaheme MtrA, and given the similarities in primary structure this could
explain why MtrA can act effectively as an electron donor for NrfA.
A key observation made in this work has been that the synthesis of MtrA
confers on E. coli the capacity to couple reduction of a range of
soluble Fe(III) species to its own respiratory electron transport chain. This
has allowed the demonstration of the capacity in vivo for reduced
MtrA as a reductase of soluble Fe(III) species. The ratio of heme to
polypeptide in MtrA is
1:28 amino acids. This compares to
1:100 in
larger multiheme enzymes such as the periplasmic nitrite reductase but is
comparable to the ratio of
1:23 in the small (
10 kDa) tetraheme
cytochromes of S. oneidensis MR-1 and S. frigidimarina
NCIMB400 for which structural data have recently emerged
(31). This arrangement of
closely packed solvent-exposed hemes has been described as one that allows for
"electron harvesting." Essentially, it might enable the protein to
be highly promiscuous in its electron donating or accepting activity with the
main driving force for electron transfer being thermodynamic. In the case of
MtrA, the observations made in this paper support this view. The decaheme
cytochrome can participate in a foreign electron transport systems in which it
can obtain electrons from the host cell inner membrane quinol dehydrogenase
and donate electrons to host cell oxidoreductases. The presence of MtrA also
allows the host cell to couple its own carbon metabolism to respiratory
Fe(III) reduction. Although MtrA has a ferric reductase activity, this is not
specific for any particular Fe(III) chelate, and the activity must arise from
electron transfer between surface-exposed hemes that come within in long range
electron transfer distance (<14 Å) of the electron accepting Fe(III)
in the chelate. This is also likely to be true for the small tetraheme
cytochromes that appear to be important for Fe(III) respiration in S.
frigidimarina (12). This
then underlies the response of Shewanella species to anaerobic growth
in the presence of soluble iron chelates, which as illustrated in
Fig. 1, is to synthesize a
large pool of multiheme periplasmic c-type cytochromes for which
there are numerous genes on the chromosome and many of which may prove to have
similar catalytic properties to MtrA.
 |
FOOTNOTES
|
---|
* This work was funded by a Biotechnology and Biological Sciences Research
Council (BBSRC) Grant P11894
[GenBank]
(to D. J. R., P. S. D., and F. R.-R.), a Wellcome
Trust Grant WT059531/2/99/2 (to H. E. S., D. J. R., and A. J. T.) and a BBSRC
research studentship (to K. P.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
||
To whom correspondence should be addressed. Tel.: 44-1603-593876; Fax:
44-1603-592003; E-mail:
h.seward{at}uea.ac.uk.
1 The abbreviations used are: Ccm, cytochrome c maturation;
Em, midpoint redox potential; NTA,
nitrilotriacetic acid; HQNO,
2-n-heptyl-4-hydroxyquinoline-N-oxide; NIR, near infrared;
Maltol, 3-hydroxy-2-methyl-4-pyrone. 
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Myles Cheesman and Dr. Julea Butt for helpful
discussions on EPR and electrochemical techniques, respectively. We also thank
Dr. Shirley Fairhurst for the additional use of a Bruker ER300 series EPR
spectrometer at the John Innes Center during this project.
 |
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