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 {ddagger}, Paul S. Dobbin §, Francisca Reyes-Ramirez {ddagger}, Andrew J. Thomson ¶, David J. Richardson {ddagger} and Harriet E. Seward {ddagger} ¶ ||

From the Centre for Metalloprotein Spectroscopy and Biology, {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 {gamma}-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 {beta}-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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Expression of mtrA in E. coli—The 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 {beta}-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 Conditions—Cells 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-{beta}-D-galactopyranoside and grown under the same conditions overnight.

Cell Fractionation and Protein Purification—Cells 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 0–250 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 550–535 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 240–800 and 700–2000 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 Titration—Mediated 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 EPR—Under a nitrogen atmosphere, mediated redox potentiometry was performed using a polished graphite electrode in the form of a carbon pot in the range of 50–273 mV. Samples were allowed to equilibrate in the carbon pot for 1–2 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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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. coli—The 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).

 

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 {alpha}-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-{beta}-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 mM–1 cm1/heme) reveals that around 6 nmol protein (mg cells)1 is produced.

Purification and Characterization of MtrA—The 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 +.

 

Optical Redox Titration—The optical spectrum of oxidized MtrA has a Soret ({gamma}) 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 {lambda}max shifts to 420 nm and {beta}- and {alpha}-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 MtrA—The 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 (800–2500 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.

 

Electron Paramagnetic Resonance of Oxidized MtrA—The 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

 

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 MtrA—A 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 MtrA—The 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 1–2 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. coli—MtrA 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 3–4, 5–6, and 7–8 are considered separately, two conserved His are found to be in an arrangement of CXXCHX10–12HX7–9CXXCHXXH. 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 CXXCHX10–12HX7–9CXXCHXXH 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. Back

|| 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. Back


    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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 ABSTRACT
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 CONCLUSIONS
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