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Electron Transfer in the Dissimilatory Iron-reducing Bacterium Geobacter metallireducens
Anaerobe (2000) 6, 187±196 doi:10.1006/anae.2000.0333
PHYSIOLOGY/STRUCTURAL BIOLOGY
ElectronTransfer in the Dissimilatory Iron-reducing Bacterium Geobacter metallireducens James E. Champine1,*, Brian Underhill1, Jamie M. Johnston1, Walt W. Lilly1, and Steve Goodwin2 1
Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701, U.S.A 2 Department of Microbiology University of Massachusetts, Amherst, Massachusetts 01003, U.S.A. (Received 6 August 1999; accepted in revised form 12 January 2000) Key Words: NADH dehydrogenase, anaerobic respiration, Geobacteraceae, Cytochrome c, Fe(III)-reduction
To investigate electron transport in the dissimilatory iron-reducing isolate Geobacter metallireducens strain GS-15, assays for redox enzymes and characterizations of cytochromes were performed. G. metallireducens produced 1.56 g dry cell weight per mol e7 transferred when grown on benzoate and contained the following citric acid cycle enzymes (activities in nkat per mg cell protein); isocitrate dehydrogenase (0.84), coenzyme A-dependent 2oxoglutarate: methyl viologen oxidoreductase (2.80), succinate dehydrogenase (0.80), and malate dehydrogenase (8.35). An oxygen-sensitive, soluble coenzyme A-dependent 2-oxoglutarate: ferredoxin oxidoreductase (0.14) with no NAD(P)-activity was observed. In cell suspensions NADPH, but not NADH, could reduce methyl viologen (2.45). Isocitrate and malate dehydrogenase activities were soluble enzymes that coupled with NADP and NAD, respectively. NADPH (0.94) and NADH (1.85) oxidation activities were observed in detergent solubilized, whole-cell suspensions using the artificial electron acceptor menadione. Menaquinone was observed at 1.2 mmol per g cell protein. The triheme c7 cytochrome was purified and 37 amino acids were determined. The mass observed by mass spectroscopy was 9684+10 Da. The average mid-point potential for the three hemes was measured at 791 mV. The growth yield, redox reactions, and electron transfer components are discussed with regards to possible sites of energy conservation during growth on iron(III). # 2000 Academic Press
Introduction Dissimilatory iron(III)-reducing bacteria are of considerable environmental and ecological interest. These *Address correspondence to: Dr. James E. Champine, Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701, Tel: +1 573 651 2385, Fax: +1 573 986 6433. E-mail: [email protected]
1075-9964/99/030187 + 10 $30.00/0
organisms are found in a wide variety of sedimentary environments [1], and iron(III)-reducers may account for a large percentage of the oxidation of organic materials in anaerobic environments [2±4]. Iron(III)reducers influence biogeochemical cycles [5] and carry out the mineralization of aromatic pollutants [6,7]. Organisms capable of Fe(III) reduction are phylogenetically diverse [8], but one group of obligately anaerobic bacteria, the Geobacteraceae, form a coherent # 2000 Academic Press
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taxon. All members of the group reduce Fe(III), while certain members can reduce elemental sulfur [8,9] and humic substances [10,11]. Fe(III), S8, and humics are interesting because they may have acted as accessory oxidants in early respiratory systems [2,10]. The reduction of iron, and the other insoluble electron acceptors, is of considerable physiological interest. The physiology of Geobacteraceae grown on Fe(III) has been investigated in two organisms; G. metallireducens and G. sulfurreducens. G. metallireducens was the first obligately anaerobic bacterium shown to use the reduction of environmentally common forms of iron to support growth [12]. The organism oxidizes various substrates completely to CO2 using the citric acid cycle [13], and transfers electron to Fe(III), presumably via menaquinone and cytochromes which have been detected in the cell [14±16]. The topography and order of electron transfer reactions has not been thoroughly characterized. Recently a model of electron transfer via a periplasmic, low-molecular-weight, triheme cytochrome c3 (albeit isolated from cells grown on fumarate) has been proposed in the closely related G. sulfurreducens [17]. The system may involve an outer membrane bound terminal oxidase [18]. Alternatively, humic substances, especially quinones, may act in the environment as a shuttle between microorganisms such as G. metallireducens and insoluble iron [10,11]. Finally, it is possible that cytochromes are not required at all to reduce Fe(III) [19]. Recently most efforts have focused on identifying the Fe(III) reductase [16±18]. Discerning how electrons are transferred to the relatively insoluble electron acceptor Fe(III) needs to be followed by basic physiological studies to determine how energy is conserved by the organism. We have used growth studies, topographical enzyme studies, protein purification, and spectrophometric analysis to develop a model for energy conservation by the organism.
Materials and Methods Organisms, growth conditions, and harvesting cell material The organisms used in this study were Geobacter metallireducens strain GS-15 and Methanosarcina barkeri MS. The medium for iron-grown G. metallireducens [FWA-Fe(III)-citrate] contained 50 mM ferric citrate and either 2 mM benzoate or 50 mM acetate [7]. The iron-reducing bacterium was grown in 100 mL liquid medium in serum bottles or 500 mL liquid medium in an anaerobic chamber as previously described [13]. For the purification of cytochrome G. metallireducens was grown in a 6 L batch culture of FWA-Fe(III)citrate medium containing 2 mM benzoate. Methanosarcina barkeri was grown in phosphate-buffered basal
medium [20] with 80 mM acetate as energy source. The anaerobic cultures were incubated statically at 308C in the dark. In one experiment, to compare growth-yield between acetate and benzoate media, the bacterium was grown in 10 mL of medium in anaerobic pressure tubes. At least five tubes were combined in each determination and four independent determinations were made for each growth substrate. The inocula used in the growth studies were 10% (v/v) from recently reduced cultures of G. metallireducens that had been grown with the same carbon source for at least five passages. Unless noted, bacteria used for enzyme assays were collected exactly as described previously [13]. Cells used for protein yield, menaquinone extraction, and protein purification were harvested under air and the pellets were stored at 7208C. Detergent-solubilized whole-cell suspensions and cell free preparations used in enzymatic studies Detergent-solubilized whole-cell suspensions were prepared exactly as previously described [13]. Detergent-to-protein ratios ranged from 2 to 10, providing enough detergent for complete lysis of the cells membranes without delipidation of protein membrane complexes [21]. To carry out cell fractionation studies, anaerobic cell pellets were suspended in extraction buffer as described by Moeller-Zinkhan and Thauer [22]. The buffer contained 50 mM Tricine/KOH, pH 7.5, with 250 mM sucrose, 20 mM KCl, 5 mM MgCl2, and 0.3 mM dithiothreitol (rather than 5 mM). The cell suspension was disrupted by sonic disintegration under continuous gassing with O2-free argon gas [23]. A Biosonik IV (VWR Scientific, San Francisco, CA, U.S.A.) sonicator was used. The suspension was subjected to three 15 s cycles (at 50% power), while the sample was kept on ice. Unbroken cells and iron precipitates were removed from the homogenote by centrifugation (48C, 10 0006g, 10 min). This supernatant, designated as cell extract, was decanted into a polyallomer UltraLok centrifuge tube (Nalgene Co., Rochester, NY, U.S.A.) in the anoxic chamber and ultracentrifuged for 1 hour (48C, 160 0006g). A fraction of the cell extract was transferred to a 12 mL serum bottle, stoppered, and held on ice until assayed. The ultracentrifuge tube was opened in the anoxic chamber and the supernatant designated as the soluble fraction. The pellet, designated as membrane fraction, was re-suspended in extraction buffer and kept under the same conditions as the previous fractions. All three fractions were subjected to several cycles of evacuation and flushing with O2-free
ElectronTransfer in Geobacter metallireducens nitrogen gas. Generally assays were performed within 4 h of preparation and never more than 12 h. This was true for detergent-solubilized, whole cell suspensions as well. Assay for CO dehydrogenase activity was done within 1 h. Enzyme assays General procedures and instruments for enzyme assays were the same as previously described [13]. Dehydrogenases (DH); isocitrate DH, 2-oxoglutarate: methyl viologen oxidoreductase, succinate DH, malate DH, and carbon monoxide DH were determined precisely as previously reported [13]. NADH and NADPH oxidase were assayed after Moeller-Zinkhan and Thauer [22]. Oxidation of the reduced nucleotides was followed in a reaction mixture containing 100 mM Ttricine/KOH, pH 7.5, 0.32 mM NADH or NADPH, 5 mM MgCl2, and 0.13 mM 2,6-dichlorophenolindophenol (DCPIP), or 0.4 mM menadione (40 mM stock solution in ethanol). NADH- and NADPH-dependent methyl viologen and ferredoxin reduction was also assayed using 0.63 and 0.54 mM of either electron donor in place of 2-oxoglutarate in the assay described previously [13]. Succinate DH was also tested with DCPIP and menadione. Fractionation of cell cytochromes and purification of cytochrome c7 About 0.6 g wet weight of G. metallireducens was resuspended in extraction buffer and subjected to the fractionation procedure described above. DNase I, 2000 Kunitz units, was included, and the sonicated lysate was kept at 358C for one hour. The following steps were carried out at 58C. The soluble fraction was dialyzed (molecular weight cut off 1 kDa) against 750 mL 50 mM Tricine/KOH buffer, pH 7.5, and diluted to 25 mL with that buffer. The sample was then applied to a 3-mL column of DEAE-cellulose (DE 52, Sigma Chemical Co., St. Louis, MO, U.S.A.) equilibrated with the same buffer, and washed with one column volume of buffer. The resulting fraction is described as DEAE effluent, and was applied to a 3mL CM-cellulose column equilibrated with the Tricine buffer. The resulting effluent is referred to as the CM effluent. A cytochrome was eluted from the CM-cellulose column with a linear gradient (20620 mL) of 0±0.5 M NaCl in Tricine buffer. The absorbance at 408 and 280 nm was measured in each fraction, the three fractions with A408 greater than 0.2 were pooled, and are referred to as cytochrome c7. The conductivity of the peak fraction was measured on a Bio-Rad Econo Gradient Monitor (Bio-Rad Laboratories, Richmond, CA, U.S.A.).
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Spectrophotometric assay for cytochromes Absorbance and difference spectra were performed on a Lambda 4B UV/Vis spectrophotometer (model no. C688-0001; Perkin Elmer, Norwalk, CT, U.S.A.). The distribution and enrichment for cytochrome was followed by the absorbance at 408 nm per mg protein under air-oxidized conditions. Dithionite-reduced minus air-oxidized difference spectra were performed by reducing one sample with excess sodium dithionite. Pyridine haemochrome assay [24] was used to quantify haem c. Reduced minus oxidized spectra were obtained using 0.8 mL sample, 0.1 mL pyridine, and 0.1 mL 5 M NaOH, with and without dithionite, and the increase in a-absorbance maximum was calculated from [(A549.57A535)reduced7(A549.57 A535)oxidized]. The haem c content was then calculated from a standard curve using horse heart cytochrome c. Additionally the purity index of Horio and Kamen [25], (A5527A570)reduced/(A280)oxidized, was used to monitor purification. These assays were carried out with 0.2 mg per mL of protein for cell extract, membrane, and soluble fractions. Reduced-plus-CO minus reduced spectrum, also called CO-difference spectrum, was obtained by subjecting the purified cytochrome (10 mg per mL) to 2 min sparging with O2free carbon monoxide in the dark. Ferrous sulfate, NADH, and NADPH were also tried as reductants at 1 mM final concentration. All measurements were done at 258C, and measurements at 280 nm were done in quartz cuvettes. Redox potentiometry Redox potentiometry of the purified cytochrome was performed as outlined by Dutton [26] under O2-free argon gas. Incremental oxidation and reduction was carried out by microlitre addition of potassium ferricyanide and sodium dithionite respectively. Mediators used in the study were phenosafranin (5 mM), phenazine methosulfate (PMS; 5 mM), phenazine ethosulfate (5 mM), 2-hydroxyl-1, 4-napthoquinone (14 mM), benzyl viologen (5 mM), and methyl viologen (MV; 12 mM). Other analytical methods Assay for quinones was performed as previously described [15]. Colorimetric determination of protein was performed by the standard Bio-Rad assay (BioRad Laboratories, Richmond, CA, U.S.A.). Chicken egg white lysozyme was used as a standard. For the protein concentration of purified cytochrome c7, horse cytochrome c was used as standard, because cytochromes tend to give higher than average absorbance
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during this modification of the Bradford assay [27]. SDS PAGE was carried out by the method of Laemmli [28] using the Mini-Protean II system as per the recommendation of the manufacturer (Bio-Rad Laboratories, Richmond, CA, U.S.A.). Duplicate gels were run, and one was stained with silver nitrate. In the second gel, b-mercaptoethanol was omitted from the sample buffer, and the gel was stained for haem [29]. The gel was immersed in 1.25 mM 3,3'-5,5'-tetramethylbenzidine in 30:70 methanol/0.25 M sodium acetate, pH 5. H2O2 was added to give 25 mM and haem-proteins appeared dark blue. The purified cytochrome was dialyzed twice against distilled water and two aliquots containing 250 picomoles were lyophilized. One sample was subjected to N-terminal sequencing on an Applied Biosystems 473A Protein Sequencer using standard Edman Chemistry. The other sample was analysed by matrix-assisted laser desorption ionization-time of flight (MALDI/TOF) spectrophotometry with a linear detector. The carrier was 1:1 H2O:Acetonitrile. Both analyses were carried out by Macomolecular Resources, Colorado State University, Fort Collins, CO, U.S.A. The protein sequence data reported in this paper will appear in the SWISS-PROT Protein Data Bank under the accession number P81894.
Results Growth of Geobacter metallireducens on benzoic acid and ferric citrate In benzoate-grown G. metallireducens, an average of 38.8 mg of cell protein (n = 4, s = 8.3) was recovered
from each litre of medium after all 50 mmol of iron had been reduced. Assuming the cells are 50% protein, a growth yield of 1.56 g dry cell weight per mol iron reduced is obtained. This is equal to the yield of protein per electron transferred, because a single electron is involved in the reduction. The yield in grams of dry cell weight per mole e7 transferred was 0.56 for acetate. Growth on benzoate produced 2.8 times as much protein per electron transferred to iron when compared with growth on acetate. Citric acid cycle dehydrogenases in benzoate-grown G. metallireducens Benzoate-grown G. metallireducens contained the enzyme activities shown in Table 1. These values are nearly identical to those observed for acetate-grown cell suspensions previously reported [13]. One notable exception is that the 2-oxoglutarate:methyl viologen oxidoreductase activity in benzoate-grown cells is 3.3 times higher than observed in acetate-grown cells. The localization of these enzyme activities, determined separately from whole-cell studies, is also given in Table 1. In solubilized whole cells, no 2oxoglutarate:methyl viologen oxidoreductase activity was seen in the absence of coenzyme A. The apparent Km for CoA was 18 mM. On the other hand, thiamine pyrophosphate could be omitted from the assay mixture without loss of activity. Cell suspensions prepared under oxic conditions had only 10% of the activity of anoxically prepared cell suspensions. When NAD+ and NADP+ were substituted for methyl viologen, no activity was seen. When clostridial
Table 1. Activities of enzymes in solubilized, whole-cell suspensions and cell-free preparations of G. metallireducens Specific activity (nkat/mg protein) observed in the following preparations: Enzyme b
Isocitrate DH 2-oxoglutarate DH Succinate DH Malate DH NADPH DH NADH DH
a
Coenzyme or artificial dye
Detergent-solubilizeda whole cells
Cell-free extract
Membrane fraction
Soluble fraction
NADP MVc Fde DCPIPg DCPIP+PMSh NAD Menadione MV DCPIP Menadione MV
0.84 2.80 0.14 0.29 0.80 8.35 0.94 2.45 2.19 1.85 0.00i
1.49 1.67 NDf ND 0.58 6.97 ND ND ND ND ND
0.05 0.00d ND ND 1.54 1.42 ND ND ND ND ND
2.61 3.86 ND ND 0.14 14.16 ND ND ND ND ND
Determined in separate experiments from cell-free extracts. DH, Dehydrogenase. c MV, Methyl viologen. d Measured under the same conditions as cell-free extract and soluble fraction. e Fd, Clostridial ferredoxin. f ND, not determined. g DCPIP, 2,6-Dichlorophenolindophenol. h PMS, Phenazine methosulfate. i Measured under the same conditions as NADPH dehydrogenase. b
ElectronTransfer in Geobacter metallireducens ferredoxin was tried as electron acceptor for 2oxoglutarate, the added ferredoxin was reduced. Unlike other citric acid cycle dehydrogenases in G. metallireducens, succinate dehydrogenase is membrane associated. Succinate-dependent rates of reduction of DCPIP and DCPIP+PMS were 0.33 and 0.77 nkat/mg cell protein, respectively, when cell suspensions were assayed under air. No carbon monoxide-dependent methyl viologen reduction was seen in benzoate-grown cell suspensions that contained 2-oxoglutarate:methyl viologen oxidoreductase activity. Specific activity of CO:MV oxidoreductase measured in Methanosarcina barkeri used as control was 3.32 nkat/mg of cell protein in this experiment. NADH and NADPH dehydrogenases in benzoate grown G. metallireducens have been observed (Table 1). The NADH dehydrogenase activity is seen to couple with DCPIP and menadione, but not with methyl viologen. NADH dehydrogenase activity, observed under air, was less than 50% of that seen under anoxic conditions. NADPH dehydrogenase is distinct from the NADH dehydrogenase in its ability to couple with the lower potential electron acceptor methyl viologen. NADPH dehydrogenase activity, observed under air, was 34% of that seen under anoxic conditions when menadione was the electron acceptor. Ferric iron-grown cells contained 1.2 mmol menaquinone per gram cell protein when benzoate was the energy source. No indication of ubiquinone was observed when extracts were subjected to thin layer chromatography on silica gel G (data not shown). Cytochromes in Geobacter metallireducens Quantitative measures of cytochrome in sub-cellular fraction of benzoate-grown cells are given in Table 2. The visible spectrum of G. metallireducens cell extract shows a single absorbance maximum at 406 nm. Dithionite-reduced minus air-oxidized difference spectra of benzoate-grown cell extract are consistent with cytochrome c maxima at 552 and 420 nm. The absorbance and difference spectra of extract, membranes, and cytoplasm are consistent with cytochromes c. The protohaem to haem c ratio, 0.37,
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suggests that only small amounts of non-c are associated with the cell. The haem c content is divided equally between the soluble and membrane fractions (see Table 2). On a protein-specific basis, the membranes are only slightly richer in haem c than the cytoplasm. The difference absorbance maxima were 422 and 552 in the membrane preparation. Purification of Geobacter metallireducens cytochrome c7 Highly purified cytochrome was eluted as an isolated protein peak from the CM column at 0.2 M NaCl or approximately 500 mS conductivity. The isolated cytochrome represented 0.1% of the cell protein and was purified almost 100 fold as based on the nmol haem c per mg protein ratio (Table 2). Measures of cytochrome purity: A408/A280 of 11.3; purity index of 2.5; and 230 nmol haem c per mg protein are above the values considered as indicative of purified cytochromes. For comparison the same parameters for commercially prepared horse heart cytochrome c are: A408/A280 of 4.5; purity index of 1.2; and 81 nmol haem c per mg protein. Electrophoresis of the purified cytochrome revealed a single band when stained for total protein with silver, and a single haemoprotein when stained with 3,3'-5,5'-tetramethylbenzidine. Properties of Geobacter metallireducens cytochrome c7 The spectrophotometric and electrophoretic properties all indicate the protein to be a c-type cytochrome. The purified cytochrome has absorbance maxima in the oxidized state at 408, 528, 351 nm, and difference maxima at 552, 522, and 481 nm. The CO difference spectrum of the protein shows a mirror image of the reduced minus oxidized spectrum. Under these conditions, the ratio (g peak 7 g trough)/(a peak7a trough) of 4.0, and the a peak at 405 nm are all consistent with a low spin c-type cytochrome [30]. Haem-specific staining of the molecule indicates thioester-bound c-type haem. The high haem content per mg protein indicated a multihaem cytochrome. Analysis of the intact protein by MALDI/TOF revealed a single peak with mass
Table 2. Quantitative measures of cytochrome preparations from G. metallireducens Purification step
Protein, mg
Emg/mL)71 408 nm
Haem c nmol
nmol Haem c per mg pro
Cell extract Membrane fraction Soluble fraction DEAE effluent CM effluent Cytochrome c7
45.3 19.5 36.1 1.01 0.117 0.052
0.84 1.25 0.32 3.78 4.07 24.3
107.1 40.8 66.3 42.7 4.1 12.0
2.4 2.1 1.8 32.9 36.5 230.4
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(m/z) of 9.68+0.01 kDa. This further attests to the homogeneity of the preparation and can be used to calculate 2.3 haem per polypeptide. N-terminal sequencing of the first 37 amino acid residues indicates homology with the c7 cytochromes of G. sulfurreducens, Desulfuromonas acetoxidans [31], and a previously published sequence [16] of the G. metallireducens protein (Figure 1). The haem 1 binding site is indicated in a similar position as the D. acetoxidans protein, and three identities are histidines which act as coordinating ligands in the c7 cytochrome of D. acetoxidans. The sequenced region has a pI of 11.1 and mean hydropathic moment of 71.44 [32]. There are no hydrophobic regions. There is no trace of this protein in samples of the membrane fraction analysed by haem-staining (data not shown). To assess the possible role of this cytochrome in electron transport reactions, the midpoint redox potential was determined. The reversible mid-point of the redox titration of the purified protein is 791 mV (Figure 2). The G. metallireducens cytochrome was reducible by sodium dithionite, but not ferrous sulfate, NADH, or NADPH. Horse heart cytochrome c was reduced by all but ferrous sulfate.
Discussion and Conclusions Growth yield of G. metallireducens and a model for energy conservation during growth on iron The observed growth yield per electron transferred, Ye- (gram dry cell weight per electron transferred), can
be used to infer the theoretical energy yield, g (mole ATP formed per mol of substrate consumed) for a given electron donor [34]. When grown on acetate and iron, the yield of G. metallireducens was approximately 0.56 g dry cell weight per mol e7 transferred. This is similar to the yield of D. acetoxidans grown on acetate and sulfur (0.53 g dry cell weight per mol e7 transferred), and D. postgatei grown on acetate and sulfate (0.60 g dry cell weight per mol e7 transferred). The theoretical energy yield, mol ATP per mol acetate consumed, is suggested to be 0.3±0.6 in these anaerobes [35]. Therefore it is reasonable to suggest that the theoretical energy yield for G. metallireducens is 0.3±0.6 mol ATP per acetate consumed. This is offered as a starting point for discussion of the overall energy gain when the oxidation of acetate is coupled to the reduction of iron by G. metallireducens. The results in this study suggest the transfer to electrons from intermediates of the citric acid cycle as shown in Figure 3. The evidence that 2-oxoglutarate is oxidized at the expense of ferredoxin is that cells contain soluble 2-OG:MV oxidoreductase activity, and can reduce clostridial ferredoxin with 2-OG. 2-Oxoacid oxidoreductases using ferredoxin as electron acceptor are found in numerous bacteria [22,36±39], including the closely related sulfur- and iron-reducing organism Desulfuromonas acetoxidans [35]. The transfer of electrons from NADPH (but not NADH) to methyl viologen is also seen, suggesting ferredoxin is re-oxidized at the expense of NADP. The primary electron acceptor for malate is NADH and for isocitrate is NADPH. None of these electron transfer reactions are
Figure 1. N-terminal sequences of G. metallireducens, G. sulfurreducens, and D. acetoxidans cytochromes c7. Amino acids found in Geobacter and Desulfuromonas proteins are in boldface, and residues that are found in only Geobacter proteins are italicized. Boxes indicate amino acid residues involved in haem binding or coordination. H1 refers to histidines involved in coordinating haem 1, while H3 refers to histidine involved in coordinating haem 3.
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Figure 2. Nernst plot of G. metallireducens cytochrome c7. Fraction of cytochrome in reduced state (&) is calculated from the net a-peak height as a fraction of the maximum net a-peak height [33]. The ratio was corrected for the absorbance due to the mediators. The dotted line is a simulation of three identical haems, n = 1, and identical Em,7 = 791 mV.
likely to be involved with the conservation of energy because of the small separation in mid-point potential between the electron donating couple and the electron accepting couple. Succinate dehydrogenase does not couple with menadione in G. metallireducens, even when phenazine methosulfate is included in the assay. This activity is carried out by an ATP-consuming succinate:menaquinone oxidoreductase in D. acetoxidans [40], suggesting this might be an energetic drain on the growth of G. metallireducens. G. metallireducens most likely conserves energy during the transfer of electrons from NAD(P)H to menaquinone (Figure 3). The levels of menaquinone reported here are similar to those previously demonstrated in the bacterium [15]. Both NADH and NADPH were oxidized in the presence of menadione, an analogue of menaquinone. Similar oxygen sensitive NAD(P)H dehydrogenase activities have been observed in other anaerobic members of the dproteobacteria; Desulfobacter postgatei [41] and several Desulfovibrio strains [42]. The separation in midpoint potential (7230 mV) is large enough to support the conservation of free energy [43]. Since G. metallireducens contains no ubiquinone it is difficult to compare its NADH oxidizing system to complex I of the mitochondrial paradigm [44]. Rotenone has been shown to be an inhibitor of NADHdependent Fe(III) reductase of G. metallireducens [14] and G. sulfurreducens [18] respectively, so there may be some biochemical similarity in the NADH oxidizing systems of these anaerobes and the rotenone-sensitive complex I of mitochondria. Mitochondrial complexes II and III contain b-type cytochromes, and complex IV contains cytochromes a. In this report only c-type cytochromes are indicated, which is consistent with every other study describing cytochromes in the Geobacteraceae [1,14,17,18,45±47]. To infer the fate of electrons beyond menaquinone, the variety and distribution of cytochromes in G. metallireducens must be considered.
Figure 3. Model for electron transfer from intermediates of the citric acid cycle to Fe(III) in Geobacter metallireducens. Non-standard abbreviations used: 2-OG, 2-oxoglutarate; Su, succinate; Fd, ferredoxin; Ictr, isocitrate; Mal, malate; OA, oxaloacetate; Mk, menaquinone; Cyt c7, cytochrome c7. The redox couples are placed vertically according to their standard reduction potentials except for the terminal oxidase whose midpoint potential was not reported and the Fe3+/Fe2+ couple which is placed according to the estimated physiological midpoint potential. Localization of reactions or intermediates, where inferred from data or as previously reported (see Discussion and Conclusions), are indicated in italics. Possible sites of energy conservation (mH+?) and ATP-consuming reactions (7ATP) are indicated.
Our results (see Table 2) show that cytochrome c is found in both the soluble and membrane fractions of G. metallireducens. Cytochrome c [17] and cytochrome containing Fe(III)-reductase [18] are found in the membrane fraction of G. sulfurreducens. The latter is associated with the outer membrane of the organism. The localization of the G. metallireducens cytochrome c to inner and outer membrane fractions could not be ascertained in the manner we carried out our experiments. This makes the role of cytoplasmic membrane-bound cytochrome in succinate, NAD(P)H, or menaquinone oxidation unclear vis-aÁvis mitochondrial complexes II and III. Geobacter cytochrome c7 described here (see below) and elsewhere [16,17] has a low mid-point potential, and may act to transfer electrons from menaquinone to a terminal oxidase in the outer membrane. The soluble cytochrome c7 is not reduced directly with NADH. The mid-point potential of cytochrome c7 (791 mV) in G. metallireducens is similar to that of menaquinone (774 mV [43]) suggesting that electron
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transfer would be favourable if the cytochrome could be rapidly re-oxidized. The failure of the protein to couple with ferrous sulfate suggests it is not directly oxidized by Fe(III). The periplasmic location is consistent with re-oxidation by a terminal oxidase present in the outer membrane as described in G. sulfurreducens [18]. The Fe3+/Fe2+ couple has a midpoint potential of +770 mV at pH 0, but under physiological ironreducing conditions, i.e. neutral pH in the presence of HCO37, the mid-point potential of the Fe(OH)3+HCO37/FeCO3 couple would be +200 mV [48]. Any terminal oxidase would have to have a more negative midpoint potential. The separation in mid-point potential between either menaquinone (774 mV) or the cytochrome c7 ( 791 mV) and the Fe3+/Fe2+ couple (near +200 mV) may be large enough to support the conservation of energy. However it is not clear how proton translocation from the cytoplasm would take place with this external oxidation system and requires further study. Characteristics of the cytochrome c7 in Geobacter metallireducens Based on the absorbance maxima, trihaem content, small size, and low redox potential the protein described here is the same as described recently by Afkar and Fukumori [16]. We had first described a trihaem protein in G. metallireducens in 1992 [49], and would like to extend the known properties of the cytochrome, especially with regards to amino acid sequence data. The mass, measured accurately by MALDI/TOF, of 9.68+0.01 kDa, is in agreement to the value of 9.5 kDa reported from SDS PAGE analysis [16], but not close to the conflicting mass of 12.0 kDa calculated from the amino acid composition given [16]. It is likely to be one amino acid larger than the G. sulfurreducens protein [17] which has a mass of 9.57 +0.02 kDa. The overall midpoint potential is measured at 791 mV, considerably more positive than the 7190 mV reported previously [16]. Our titration used a much wider range of redox potentials than used to study the cytochromes from either G. metallireducens [16] or G. sulfurreducens [17]. The non-sigmoidal shape of the redox titration indicates non-equivalent mid-point potentials for the haems, which is expected if the redox centers are not in identical amino acid environments [50]. This is true for the mid-point potentials of the haems in cytochrome c7 from D. acetoxidans, which were measured as 7102, 7177, and 7177 mV [51]. The yield of purified cytochrome, 0.1%, can be used to estimate the number of polypeptides in an average cell. Assuming a protein content similar to Escherichia coli of
1.5610715 g per cell [52], and a molecular weight of 9684, there are approximately 10 000 copies of this protein per cell. This would be ample cell associated protein to facilitate electron transport. Consideration of the amino acid sequence of the protein (Figure 1), including new sequence data, can be used to infer additional properties of the cytochrome. Previously described cytochrome c7 proteins have been localized to the periplasm [17]. The G. metallireducens c7 has an N-terminus of Ala-Asp indicative of a signal peptide cleavage site [53], and therefore supports the hypothesis that this is a periplasmic protein. The partial sequence data indicates no hydrophobic regions, so unlike the protein described in G. sulfurreducens [17], there is no evidence for membrane association. Also, that protein is described as having a near-neutral isoelectric point, while the sequence data for the G. metallireducens protein indicates a very basic protein (pI > 10). The basic nature of the G. metallireducens protein is further supported by its binding to carboxymethyl-cellulose, and the lysine-rich content [16]. It is unlikely that this positively charged hydrophilic protein would be membrane associated. Amino acid sequence data provides a molecular basis for the assignment of the G. metallireducens protein as a cytochrome c7. The haem 1 binding site of the D. acetoxidans protein is Cys-Asp-Ala-Cys-His (residues 26 to 30 of the mature D. acetoxidans protein sequence) and the additional His ligand is at amino acid 17 [31]. Residues 27 to 31, Cys-Lys-Lys-Cys-His in the G. metallireducens resembles the haem 1 binding site in the D. acetoxidans protein. Both have in common the His at position number 17, as well as a His at position number 20 which is the accessory ligand for haem 3 in the D. acetoxidans protein. Class III cytochromes have bis-histidinyl coordination, and therefore at least six histidines are expected. Afkar and Fukumori [16] report only four, while D. acetoxidans has six [54] and we estimate as many as ten in the G. metallireducens protein (data not shown). These similarities, and other amino acid identities suggest the gene that codes for the G. metallireducens protein is of the same gene family that codes for c7 and c3 cytochromes. Trihaem cytochromes c7 are homologous to the c3 cytochrome of Desulfovibrio without the haem 2 of the tetrahaem protein [31]. The presence of this cytochrome extends the recognized physiological similarities between the genera Geobacter and Desulfuromonas. These properties include an unusual, low-potential 2-oxoglutarate dehydrogenase [13] and the presence of menaquinone rather than ubiquinone [15]. Also, the documentation of these c7 cytochromes in iron-, humics-, and sulfurreducing anaerobes may help elucidate the mechanism of electron transport to these insoluble substrates.
ElectronTransfer in Geobacter metallireducens Acknowledgments This work was supported by a grant from the Office of Naval Research, Grant # N00014-91-J-1898, and a grant from the Grants and Research Funding Committee, Southeast Missouri State University. We would like to thank Ed Towers and Phil Ryan for technical assistance with the amino acid sequence analysis and Derek Lovley and John Stolz for useful discussion.
References 1. Coates J.D., Phillips E.J.P., Lonergan D.J., Jenter H.L. and Lovley D.R. (1996) Isolation of Geobacter species from diverse sedimentary environments. Appl Environ Microbiol 62: 1531±1536 2. Lovely D.R. (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev 55: 259±287 3. Lovely D.R. (1997) Microbial Fe(III) reduction in subsurface environments. FEMS Microbiol Lett 20: 305±313 4. Ponnamperuma F.N. (1972) The chemistry of submerged soils. Adv Agron 24: 29±96 5. Nealson K.H. and Saffarini D. (1994) Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation. Ann Rev Microbiol 48: 311±343 6. Lovley D.R., Baedecker M.J., Lonergan, D.J., Cozzarelli I.M., Phillips E.J.P. and Siegel D.I. (1989) Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339: 297±299 7. Lovley D.R. and Lonergan D.J. (1990) Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GS-15. Appl Environ Microbiol 56: 1858±1864 8. Lonergan D.J., Jenter H.L., Coates J.D., Phillips E.J.P., Schmidt T.M. and Lovley D.R. (1996) Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. J Bacteriol 178: 2402±2408 9. Roden E.E. and Lovley D.R. (1993) Dissimilatory Fe(III) reduction by the marine microorganism Desulfuromonas acetoxidans. Appl Environ Microbiol 59: 734±742 10. Lovley D.R., Coates J.D., Blunt-Harris E.L., Phillips E.J.P. and Woodward J.C. (1996) Humic substances as electron acceptors for microbial respiration. Nature 382: 445±448 11. Scott D.T., McKnight D.M., Blunt-Harris E.L., Kolesar S.E. and Lovley D.R. (1998) Quinone moieties act as electron acceptors in the reduction of humic substances by humics-reducing microorganisms. Environ Sci Technol 32: 2984±2989 12. Lovley D.R., Stolz J.F., Nord G.L. and Phillips E.J.P. (1987) Anaerobic production of magnetite by a dissimilatory ironreducing microorganism. Nature 330: 252±254 13. Champine J.E. and Goodwin S. (1991) Acetate catabolism in the dissimilatory iron-reducing isolate GS-15. J Bacteriol 173: 2704± 2706 14. Gorby Y.A. and Lovley D.R. (1991) Electron transport in the dissimilatory iron reducer, GS-15. Appl Environ Microbiol 57: 867±870 15. Lovley D.R., Giovannoni S.J., White D.C., Champine J.E., Phillips E.J.P., Gorby Y.A. and Goodwin S. (1993) Geobacter metallireducens gen nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other minerals. Arch Microbiol 159: 336± 344 16. Afkar E. and Fukumori Y. (1999) Purification and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens. FEMS Microbiol Lett 175: 205±210 17. Seeliger S., Cord-Ruwisch R. and Schink B. (1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as electron carrier to other acceptors or to partner bacteria. J Bacteriol 180: 3686±3691 18. Gaspard S., Vazquez F. and Holliger C. (1998) Localization and solubilization of the iron(III) reductase of Geobacter sulfurreducens. Appl Environ Microbiol 64: 3188±3194. 19. Lovley D.R., Phillips E.J.P., Lonergan D.J. and Widman P.K. (1995) Fe(III) and S8 reduction by Pelobacter carbinolicus. Appl Environ Microbiol 61: 2132±2138
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PHYSIOLOGY/STRUCTURAL BIOLOGY
ElectronTransfer in the Dissimilatory Iron-reducing Bacterium Geobacter metallireducens James E. Champine1,*, Brian Underhill1, Jamie M. Johnston1, Walt W. Lilly1, and Steve Goodwin2 1
Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701, U.S.A 2 Department of Microbiology University of Massachusetts, Amherst, Massachusetts 01003, U.S.A. (Received 6 August 1999; accepted in revised form 12 January 2000) Key Words: NADH dehydrogenase, anaerobic respiration, Geobacteraceae, Cytochrome c, Fe(III)-reduction
To investigate electron transport in the dissimilatory iron-reducing isolate Geobacter metallireducens strain GS-15, assays for redox enzymes and characterizations of cytochromes were performed. G. metallireducens produced 1.56 g dry cell weight per mol e7 transferred when grown on benzoate and contained the following citric acid cycle enzymes (activities in nkat per mg cell protein); isocitrate dehydrogenase (0.84), coenzyme A-dependent 2oxoglutarate: methyl viologen oxidoreductase (2.80), succinate dehydrogenase (0.80), and malate dehydrogenase (8.35). An oxygen-sensitive, soluble coenzyme A-dependent 2-oxoglutarate: ferredoxin oxidoreductase (0.14) with no NAD(P)-activity was observed. In cell suspensions NADPH, but not NADH, could reduce methyl viologen (2.45). Isocitrate and malate dehydrogenase activities were soluble enzymes that coupled with NADP and NAD, respectively. NADPH (0.94) and NADH (1.85) oxidation activities were observed in detergent solubilized, whole-cell suspensions using the artificial electron acceptor menadione. Menaquinone was observed at 1.2 mmol per g cell protein. The triheme c7 cytochrome was purified and 37 amino acids were determined. The mass observed by mass spectroscopy was 9684+10 Da. The average mid-point potential for the three hemes was measured at 791 mV. The growth yield, redox reactions, and electron transfer components are discussed with regards to possible sites of energy conservation during growth on iron(III). # 2000 Academic Press
Introduction Dissimilatory iron(III)-reducing bacteria are of considerable environmental and ecological interest. These *Address correspondence to: Dr. James E. Champine, Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701, Tel: +1 573 651 2385, Fax: +1 573 986 6433. E-mail: [email protected]
1075-9964/99/030187 + 10 $30.00/0
organisms are found in a wide variety of sedimentary environments [1], and iron(III)-reducers may account for a large percentage of the oxidation of organic materials in anaerobic environments [2±4]. Iron(III)reducers influence biogeochemical cycles [5] and carry out the mineralization of aromatic pollutants [6,7]. Organisms capable of Fe(III) reduction are phylogenetically diverse [8], but one group of obligately anaerobic bacteria, the Geobacteraceae, form a coherent # 2000 Academic Press
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taxon. All members of the group reduce Fe(III), while certain members can reduce elemental sulfur [8,9] and humic substances [10,11]. Fe(III), S8, and humics are interesting because they may have acted as accessory oxidants in early respiratory systems [2,10]. The reduction of iron, and the other insoluble electron acceptors, is of considerable physiological interest. The physiology of Geobacteraceae grown on Fe(III) has been investigated in two organisms; G. metallireducens and G. sulfurreducens. G. metallireducens was the first obligately anaerobic bacterium shown to use the reduction of environmentally common forms of iron to support growth [12]. The organism oxidizes various substrates completely to CO2 using the citric acid cycle [13], and transfers electron to Fe(III), presumably via menaquinone and cytochromes which have been detected in the cell [14±16]. The topography and order of electron transfer reactions has not been thoroughly characterized. Recently a model of electron transfer via a periplasmic, low-molecular-weight, triheme cytochrome c3 (albeit isolated from cells grown on fumarate) has been proposed in the closely related G. sulfurreducens [17]. The system may involve an outer membrane bound terminal oxidase [18]. Alternatively, humic substances, especially quinones, may act in the environment as a shuttle between microorganisms such as G. metallireducens and insoluble iron [10,11]. Finally, it is possible that cytochromes are not required at all to reduce Fe(III) [19]. Recently most efforts have focused on identifying the Fe(III) reductase [16±18]. Discerning how electrons are transferred to the relatively insoluble electron acceptor Fe(III) needs to be followed by basic physiological studies to determine how energy is conserved by the organism. We have used growth studies, topographical enzyme studies, protein purification, and spectrophometric analysis to develop a model for energy conservation by the organism.
Materials and Methods Organisms, growth conditions, and harvesting cell material The organisms used in this study were Geobacter metallireducens strain GS-15 and Methanosarcina barkeri MS. The medium for iron-grown G. metallireducens [FWA-Fe(III)-citrate] contained 50 mM ferric citrate and either 2 mM benzoate or 50 mM acetate [7]. The iron-reducing bacterium was grown in 100 mL liquid medium in serum bottles or 500 mL liquid medium in an anaerobic chamber as previously described [13]. For the purification of cytochrome G. metallireducens was grown in a 6 L batch culture of FWA-Fe(III)citrate medium containing 2 mM benzoate. Methanosarcina barkeri was grown in phosphate-buffered basal
medium [20] with 80 mM acetate as energy source. The anaerobic cultures were incubated statically at 308C in the dark. In one experiment, to compare growth-yield between acetate and benzoate media, the bacterium was grown in 10 mL of medium in anaerobic pressure tubes. At least five tubes were combined in each determination and four independent determinations were made for each growth substrate. The inocula used in the growth studies were 10% (v/v) from recently reduced cultures of G. metallireducens that had been grown with the same carbon source for at least five passages. Unless noted, bacteria used for enzyme assays were collected exactly as described previously [13]. Cells used for protein yield, menaquinone extraction, and protein purification were harvested under air and the pellets were stored at 7208C. Detergent-solubilized whole-cell suspensions and cell free preparations used in enzymatic studies Detergent-solubilized whole-cell suspensions were prepared exactly as previously described [13]. Detergent-to-protein ratios ranged from 2 to 10, providing enough detergent for complete lysis of the cells membranes without delipidation of protein membrane complexes [21]. To carry out cell fractionation studies, anaerobic cell pellets were suspended in extraction buffer as described by Moeller-Zinkhan and Thauer [22]. The buffer contained 50 mM Tricine/KOH, pH 7.5, with 250 mM sucrose, 20 mM KCl, 5 mM MgCl2, and 0.3 mM dithiothreitol (rather than 5 mM). The cell suspension was disrupted by sonic disintegration under continuous gassing with O2-free argon gas [23]. A Biosonik IV (VWR Scientific, San Francisco, CA, U.S.A.) sonicator was used. The suspension was subjected to three 15 s cycles (at 50% power), while the sample was kept on ice. Unbroken cells and iron precipitates were removed from the homogenote by centrifugation (48C, 10 0006g, 10 min). This supernatant, designated as cell extract, was decanted into a polyallomer UltraLok centrifuge tube (Nalgene Co., Rochester, NY, U.S.A.) in the anoxic chamber and ultracentrifuged for 1 hour (48C, 160 0006g). A fraction of the cell extract was transferred to a 12 mL serum bottle, stoppered, and held on ice until assayed. The ultracentrifuge tube was opened in the anoxic chamber and the supernatant designated as the soluble fraction. The pellet, designated as membrane fraction, was re-suspended in extraction buffer and kept under the same conditions as the previous fractions. All three fractions were subjected to several cycles of evacuation and flushing with O2-free
ElectronTransfer in Geobacter metallireducens nitrogen gas. Generally assays were performed within 4 h of preparation and never more than 12 h. This was true for detergent-solubilized, whole cell suspensions as well. Assay for CO dehydrogenase activity was done within 1 h. Enzyme assays General procedures and instruments for enzyme assays were the same as previously described [13]. Dehydrogenases (DH); isocitrate DH, 2-oxoglutarate: methyl viologen oxidoreductase, succinate DH, malate DH, and carbon monoxide DH were determined precisely as previously reported [13]. NADH and NADPH oxidase were assayed after Moeller-Zinkhan and Thauer [22]. Oxidation of the reduced nucleotides was followed in a reaction mixture containing 100 mM Ttricine/KOH, pH 7.5, 0.32 mM NADH or NADPH, 5 mM MgCl2, and 0.13 mM 2,6-dichlorophenolindophenol (DCPIP), or 0.4 mM menadione (40 mM stock solution in ethanol). NADH- and NADPH-dependent methyl viologen and ferredoxin reduction was also assayed using 0.63 and 0.54 mM of either electron donor in place of 2-oxoglutarate in the assay described previously [13]. Succinate DH was also tested with DCPIP and menadione. Fractionation of cell cytochromes and purification of cytochrome c7 About 0.6 g wet weight of G. metallireducens was resuspended in extraction buffer and subjected to the fractionation procedure described above. DNase I, 2000 Kunitz units, was included, and the sonicated lysate was kept at 358C for one hour. The following steps were carried out at 58C. The soluble fraction was dialyzed (molecular weight cut off 1 kDa) against 750 mL 50 mM Tricine/KOH buffer, pH 7.5, and diluted to 25 mL with that buffer. The sample was then applied to a 3-mL column of DEAE-cellulose (DE 52, Sigma Chemical Co., St. Louis, MO, U.S.A.) equilibrated with the same buffer, and washed with one column volume of buffer. The resulting fraction is described as DEAE effluent, and was applied to a 3mL CM-cellulose column equilibrated with the Tricine buffer. The resulting effluent is referred to as the CM effluent. A cytochrome was eluted from the CM-cellulose column with a linear gradient (20620 mL) of 0±0.5 M NaCl in Tricine buffer. The absorbance at 408 and 280 nm was measured in each fraction, the three fractions with A408 greater than 0.2 were pooled, and are referred to as cytochrome c7. The conductivity of the peak fraction was measured on a Bio-Rad Econo Gradient Monitor (Bio-Rad Laboratories, Richmond, CA, U.S.A.).
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Spectrophotometric assay for cytochromes Absorbance and difference spectra were performed on a Lambda 4B UV/Vis spectrophotometer (model no. C688-0001; Perkin Elmer, Norwalk, CT, U.S.A.). The distribution and enrichment for cytochrome was followed by the absorbance at 408 nm per mg protein under air-oxidized conditions. Dithionite-reduced minus air-oxidized difference spectra were performed by reducing one sample with excess sodium dithionite. Pyridine haemochrome assay [24] was used to quantify haem c. Reduced minus oxidized spectra were obtained using 0.8 mL sample, 0.1 mL pyridine, and 0.1 mL 5 M NaOH, with and without dithionite, and the increase in a-absorbance maximum was calculated from [(A549.57A535)reduced7(A549.57 A535)oxidized]. The haem c content was then calculated from a standard curve using horse heart cytochrome c. Additionally the purity index of Horio and Kamen [25], (A5527A570)reduced/(A280)oxidized, was used to monitor purification. These assays were carried out with 0.2 mg per mL of protein for cell extract, membrane, and soluble fractions. Reduced-plus-CO minus reduced spectrum, also called CO-difference spectrum, was obtained by subjecting the purified cytochrome (10 mg per mL) to 2 min sparging with O2free carbon monoxide in the dark. Ferrous sulfate, NADH, and NADPH were also tried as reductants at 1 mM final concentration. All measurements were done at 258C, and measurements at 280 nm were done in quartz cuvettes. Redox potentiometry Redox potentiometry of the purified cytochrome was performed as outlined by Dutton [26] under O2-free argon gas. Incremental oxidation and reduction was carried out by microlitre addition of potassium ferricyanide and sodium dithionite respectively. Mediators used in the study were phenosafranin (5 mM), phenazine methosulfate (PMS; 5 mM), phenazine ethosulfate (5 mM), 2-hydroxyl-1, 4-napthoquinone (14 mM), benzyl viologen (5 mM), and methyl viologen (MV; 12 mM). Other analytical methods Assay for quinones was performed as previously described [15]. Colorimetric determination of protein was performed by the standard Bio-Rad assay (BioRad Laboratories, Richmond, CA, U.S.A.). Chicken egg white lysozyme was used as a standard. For the protein concentration of purified cytochrome c7, horse cytochrome c was used as standard, because cytochromes tend to give higher than average absorbance
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during this modification of the Bradford assay [27]. SDS PAGE was carried out by the method of Laemmli [28] using the Mini-Protean II system as per the recommendation of the manufacturer (Bio-Rad Laboratories, Richmond, CA, U.S.A.). Duplicate gels were run, and one was stained with silver nitrate. In the second gel, b-mercaptoethanol was omitted from the sample buffer, and the gel was stained for haem [29]. The gel was immersed in 1.25 mM 3,3'-5,5'-tetramethylbenzidine in 30:70 methanol/0.25 M sodium acetate, pH 5. H2O2 was added to give 25 mM and haem-proteins appeared dark blue. The purified cytochrome was dialyzed twice against distilled water and two aliquots containing 250 picomoles were lyophilized. One sample was subjected to N-terminal sequencing on an Applied Biosystems 473A Protein Sequencer using standard Edman Chemistry. The other sample was analysed by matrix-assisted laser desorption ionization-time of flight (MALDI/TOF) spectrophotometry with a linear detector. The carrier was 1:1 H2O:Acetonitrile. Both analyses were carried out by Macomolecular Resources, Colorado State University, Fort Collins, CO, U.S.A. The protein sequence data reported in this paper will appear in the SWISS-PROT Protein Data Bank under the accession number P81894.
Results Growth of Geobacter metallireducens on benzoic acid and ferric citrate In benzoate-grown G. metallireducens, an average of 38.8 mg of cell protein (n = 4, s = 8.3) was recovered
from each litre of medium after all 50 mmol of iron had been reduced. Assuming the cells are 50% protein, a growth yield of 1.56 g dry cell weight per mol iron reduced is obtained. This is equal to the yield of protein per electron transferred, because a single electron is involved in the reduction. The yield in grams of dry cell weight per mole e7 transferred was 0.56 for acetate. Growth on benzoate produced 2.8 times as much protein per electron transferred to iron when compared with growth on acetate. Citric acid cycle dehydrogenases in benzoate-grown G. metallireducens Benzoate-grown G. metallireducens contained the enzyme activities shown in Table 1. These values are nearly identical to those observed for acetate-grown cell suspensions previously reported [13]. One notable exception is that the 2-oxoglutarate:methyl viologen oxidoreductase activity in benzoate-grown cells is 3.3 times higher than observed in acetate-grown cells. The localization of these enzyme activities, determined separately from whole-cell studies, is also given in Table 1. In solubilized whole cells, no 2oxoglutarate:methyl viologen oxidoreductase activity was seen in the absence of coenzyme A. The apparent Km for CoA was 18 mM. On the other hand, thiamine pyrophosphate could be omitted from the assay mixture without loss of activity. Cell suspensions prepared under oxic conditions had only 10% of the activity of anoxically prepared cell suspensions. When NAD+ and NADP+ were substituted for methyl viologen, no activity was seen. When clostridial
Table 1. Activities of enzymes in solubilized, whole-cell suspensions and cell-free preparations of G. metallireducens Specific activity (nkat/mg protein) observed in the following preparations: Enzyme b
Isocitrate DH 2-oxoglutarate DH Succinate DH Malate DH NADPH DH NADH DH
a
Coenzyme or artificial dye
Detergent-solubilizeda whole cells
Cell-free extract
Membrane fraction
Soluble fraction
NADP MVc Fde DCPIPg DCPIP+PMSh NAD Menadione MV DCPIP Menadione MV
0.84 2.80 0.14 0.29 0.80 8.35 0.94 2.45 2.19 1.85 0.00i
1.49 1.67 NDf ND 0.58 6.97 ND ND ND ND ND
0.05 0.00d ND ND 1.54 1.42 ND ND ND ND ND
2.61 3.86 ND ND 0.14 14.16 ND ND ND ND ND
Determined in separate experiments from cell-free extracts. DH, Dehydrogenase. c MV, Methyl viologen. d Measured under the same conditions as cell-free extract and soluble fraction. e Fd, Clostridial ferredoxin. f ND, not determined. g DCPIP, 2,6-Dichlorophenolindophenol. h PMS, Phenazine methosulfate. i Measured under the same conditions as NADPH dehydrogenase. b
ElectronTransfer in Geobacter metallireducens ferredoxin was tried as electron acceptor for 2oxoglutarate, the added ferredoxin was reduced. Unlike other citric acid cycle dehydrogenases in G. metallireducens, succinate dehydrogenase is membrane associated. Succinate-dependent rates of reduction of DCPIP and DCPIP+PMS were 0.33 and 0.77 nkat/mg cell protein, respectively, when cell suspensions were assayed under air. No carbon monoxide-dependent methyl viologen reduction was seen in benzoate-grown cell suspensions that contained 2-oxoglutarate:methyl viologen oxidoreductase activity. Specific activity of CO:MV oxidoreductase measured in Methanosarcina barkeri used as control was 3.32 nkat/mg of cell protein in this experiment. NADH and NADPH dehydrogenases in benzoate grown G. metallireducens have been observed (Table 1). The NADH dehydrogenase activity is seen to couple with DCPIP and menadione, but not with methyl viologen. NADH dehydrogenase activity, observed under air, was less than 50% of that seen under anoxic conditions. NADPH dehydrogenase is distinct from the NADH dehydrogenase in its ability to couple with the lower potential electron acceptor methyl viologen. NADPH dehydrogenase activity, observed under air, was 34% of that seen under anoxic conditions when menadione was the electron acceptor. Ferric iron-grown cells contained 1.2 mmol menaquinone per gram cell protein when benzoate was the energy source. No indication of ubiquinone was observed when extracts were subjected to thin layer chromatography on silica gel G (data not shown). Cytochromes in Geobacter metallireducens Quantitative measures of cytochrome in sub-cellular fraction of benzoate-grown cells are given in Table 2. The visible spectrum of G. metallireducens cell extract shows a single absorbance maximum at 406 nm. Dithionite-reduced minus air-oxidized difference spectra of benzoate-grown cell extract are consistent with cytochrome c maxima at 552 and 420 nm. The absorbance and difference spectra of extract, membranes, and cytoplasm are consistent with cytochromes c. The protohaem to haem c ratio, 0.37,
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suggests that only small amounts of non-c are associated with the cell. The haem c content is divided equally between the soluble and membrane fractions (see Table 2). On a protein-specific basis, the membranes are only slightly richer in haem c than the cytoplasm. The difference absorbance maxima were 422 and 552 in the membrane preparation. Purification of Geobacter metallireducens cytochrome c7 Highly purified cytochrome was eluted as an isolated protein peak from the CM column at 0.2 M NaCl or approximately 500 mS conductivity. The isolated cytochrome represented 0.1% of the cell protein and was purified almost 100 fold as based on the nmol haem c per mg protein ratio (Table 2). Measures of cytochrome purity: A408/A280 of 11.3; purity index of 2.5; and 230 nmol haem c per mg protein are above the values considered as indicative of purified cytochromes. For comparison the same parameters for commercially prepared horse heart cytochrome c are: A408/A280 of 4.5; purity index of 1.2; and 81 nmol haem c per mg protein. Electrophoresis of the purified cytochrome revealed a single band when stained for total protein with silver, and a single haemoprotein when stained with 3,3'-5,5'-tetramethylbenzidine. Properties of Geobacter metallireducens cytochrome c7 The spectrophotometric and electrophoretic properties all indicate the protein to be a c-type cytochrome. The purified cytochrome has absorbance maxima in the oxidized state at 408, 528, 351 nm, and difference maxima at 552, 522, and 481 nm. The CO difference spectrum of the protein shows a mirror image of the reduced minus oxidized spectrum. Under these conditions, the ratio (g peak 7 g trough)/(a peak7a trough) of 4.0, and the a peak at 405 nm are all consistent with a low spin c-type cytochrome [30]. Haem-specific staining of the molecule indicates thioester-bound c-type haem. The high haem content per mg protein indicated a multihaem cytochrome. Analysis of the intact protein by MALDI/TOF revealed a single peak with mass
Table 2. Quantitative measures of cytochrome preparations from G. metallireducens Purification step
Protein, mg
Emg/mL)71 408 nm
Haem c nmol
nmol Haem c per mg pro
Cell extract Membrane fraction Soluble fraction DEAE effluent CM effluent Cytochrome c7
45.3 19.5 36.1 1.01 0.117 0.052
0.84 1.25 0.32 3.78 4.07 24.3
107.1 40.8 66.3 42.7 4.1 12.0
2.4 2.1 1.8 32.9 36.5 230.4
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(m/z) of 9.68+0.01 kDa. This further attests to the homogeneity of the preparation and can be used to calculate 2.3 haem per polypeptide. N-terminal sequencing of the first 37 amino acid residues indicates homology with the c7 cytochromes of G. sulfurreducens, Desulfuromonas acetoxidans [31], and a previously published sequence [16] of the G. metallireducens protein (Figure 1). The haem 1 binding site is indicated in a similar position as the D. acetoxidans protein, and three identities are histidines which act as coordinating ligands in the c7 cytochrome of D. acetoxidans. The sequenced region has a pI of 11.1 and mean hydropathic moment of 71.44 [32]. There are no hydrophobic regions. There is no trace of this protein in samples of the membrane fraction analysed by haem-staining (data not shown). To assess the possible role of this cytochrome in electron transport reactions, the midpoint redox potential was determined. The reversible mid-point of the redox titration of the purified protein is 791 mV (Figure 2). The G. metallireducens cytochrome was reducible by sodium dithionite, but not ferrous sulfate, NADH, or NADPH. Horse heart cytochrome c was reduced by all but ferrous sulfate.
Discussion and Conclusions Growth yield of G. metallireducens and a model for energy conservation during growth on iron The observed growth yield per electron transferred, Ye- (gram dry cell weight per electron transferred), can
be used to infer the theoretical energy yield, g (mole ATP formed per mol of substrate consumed) for a given electron donor [34]. When grown on acetate and iron, the yield of G. metallireducens was approximately 0.56 g dry cell weight per mol e7 transferred. This is similar to the yield of D. acetoxidans grown on acetate and sulfur (0.53 g dry cell weight per mol e7 transferred), and D. postgatei grown on acetate and sulfate (0.60 g dry cell weight per mol e7 transferred). The theoretical energy yield, mol ATP per mol acetate consumed, is suggested to be 0.3±0.6 in these anaerobes [35]. Therefore it is reasonable to suggest that the theoretical energy yield for G. metallireducens is 0.3±0.6 mol ATP per acetate consumed. This is offered as a starting point for discussion of the overall energy gain when the oxidation of acetate is coupled to the reduction of iron by G. metallireducens. The results in this study suggest the transfer to electrons from intermediates of the citric acid cycle as shown in Figure 3. The evidence that 2-oxoglutarate is oxidized at the expense of ferredoxin is that cells contain soluble 2-OG:MV oxidoreductase activity, and can reduce clostridial ferredoxin with 2-OG. 2-Oxoacid oxidoreductases using ferredoxin as electron acceptor are found in numerous bacteria [22,36±39], including the closely related sulfur- and iron-reducing organism Desulfuromonas acetoxidans [35]. The transfer of electrons from NADPH (but not NADH) to methyl viologen is also seen, suggesting ferredoxin is re-oxidized at the expense of NADP. The primary electron acceptor for malate is NADH and for isocitrate is NADPH. None of these electron transfer reactions are
Figure 1. N-terminal sequences of G. metallireducens, G. sulfurreducens, and D. acetoxidans cytochromes c7. Amino acids found in Geobacter and Desulfuromonas proteins are in boldface, and residues that are found in only Geobacter proteins are italicized. Boxes indicate amino acid residues involved in haem binding or coordination. H1 refers to histidines involved in coordinating haem 1, while H3 refers to histidine involved in coordinating haem 3.
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Figure 2. Nernst plot of G. metallireducens cytochrome c7. Fraction of cytochrome in reduced state (&) is calculated from the net a-peak height as a fraction of the maximum net a-peak height [33]. The ratio was corrected for the absorbance due to the mediators. The dotted line is a simulation of three identical haems, n = 1, and identical Em,7 = 791 mV.
likely to be involved with the conservation of energy because of the small separation in mid-point potential between the electron donating couple and the electron accepting couple. Succinate dehydrogenase does not couple with menadione in G. metallireducens, even when phenazine methosulfate is included in the assay. This activity is carried out by an ATP-consuming succinate:menaquinone oxidoreductase in D. acetoxidans [40], suggesting this might be an energetic drain on the growth of G. metallireducens. G. metallireducens most likely conserves energy during the transfer of electrons from NAD(P)H to menaquinone (Figure 3). The levels of menaquinone reported here are similar to those previously demonstrated in the bacterium [15]. Both NADH and NADPH were oxidized in the presence of menadione, an analogue of menaquinone. Similar oxygen sensitive NAD(P)H dehydrogenase activities have been observed in other anaerobic members of the dproteobacteria; Desulfobacter postgatei [41] and several Desulfovibrio strains [42]. The separation in midpoint potential (7230 mV) is large enough to support the conservation of free energy [43]. Since G. metallireducens contains no ubiquinone it is difficult to compare its NADH oxidizing system to complex I of the mitochondrial paradigm [44]. Rotenone has been shown to be an inhibitor of NADHdependent Fe(III) reductase of G. metallireducens [14] and G. sulfurreducens [18] respectively, so there may be some biochemical similarity in the NADH oxidizing systems of these anaerobes and the rotenone-sensitive complex I of mitochondria. Mitochondrial complexes II and III contain b-type cytochromes, and complex IV contains cytochromes a. In this report only c-type cytochromes are indicated, which is consistent with every other study describing cytochromes in the Geobacteraceae [1,14,17,18,45±47]. To infer the fate of electrons beyond menaquinone, the variety and distribution of cytochromes in G. metallireducens must be considered.
Figure 3. Model for electron transfer from intermediates of the citric acid cycle to Fe(III) in Geobacter metallireducens. Non-standard abbreviations used: 2-OG, 2-oxoglutarate; Su, succinate; Fd, ferredoxin; Ictr, isocitrate; Mal, malate; OA, oxaloacetate; Mk, menaquinone; Cyt c7, cytochrome c7. The redox couples are placed vertically according to their standard reduction potentials except for the terminal oxidase whose midpoint potential was not reported and the Fe3+/Fe2+ couple which is placed according to the estimated physiological midpoint potential. Localization of reactions or intermediates, where inferred from data or as previously reported (see Discussion and Conclusions), are indicated in italics. Possible sites of energy conservation (mH+?) and ATP-consuming reactions (7ATP) are indicated.
Our results (see Table 2) show that cytochrome c is found in both the soluble and membrane fractions of G. metallireducens. Cytochrome c [17] and cytochrome containing Fe(III)-reductase [18] are found in the membrane fraction of G. sulfurreducens. The latter is associated with the outer membrane of the organism. The localization of the G. metallireducens cytochrome c to inner and outer membrane fractions could not be ascertained in the manner we carried out our experiments. This makes the role of cytoplasmic membrane-bound cytochrome in succinate, NAD(P)H, or menaquinone oxidation unclear vis-aÁvis mitochondrial complexes II and III. Geobacter cytochrome c7 described here (see below) and elsewhere [16,17] has a low mid-point potential, and may act to transfer electrons from menaquinone to a terminal oxidase in the outer membrane. The soluble cytochrome c7 is not reduced directly with NADH. The mid-point potential of cytochrome c7 (791 mV) in G. metallireducens is similar to that of menaquinone (774 mV [43]) suggesting that electron
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transfer would be favourable if the cytochrome could be rapidly re-oxidized. The failure of the protein to couple with ferrous sulfate suggests it is not directly oxidized by Fe(III). The periplasmic location is consistent with re-oxidation by a terminal oxidase present in the outer membrane as described in G. sulfurreducens [18]. The Fe3+/Fe2+ couple has a midpoint potential of +770 mV at pH 0, but under physiological ironreducing conditions, i.e. neutral pH in the presence of HCO37, the mid-point potential of the Fe(OH)3+HCO37/FeCO3 couple would be +200 mV [48]. Any terminal oxidase would have to have a more negative midpoint potential. The separation in mid-point potential between either menaquinone (774 mV) or the cytochrome c7 ( 791 mV) and the Fe3+/Fe2+ couple (near +200 mV) may be large enough to support the conservation of energy. However it is not clear how proton translocation from the cytoplasm would take place with this external oxidation system and requires further study. Characteristics of the cytochrome c7 in Geobacter metallireducens Based on the absorbance maxima, trihaem content, small size, and low redox potential the protein described here is the same as described recently by Afkar and Fukumori [16]. We had first described a trihaem protein in G. metallireducens in 1992 [49], and would like to extend the known properties of the cytochrome, especially with regards to amino acid sequence data. The mass, measured accurately by MALDI/TOF, of 9.68+0.01 kDa, is in agreement to the value of 9.5 kDa reported from SDS PAGE analysis [16], but not close to the conflicting mass of 12.0 kDa calculated from the amino acid composition given [16]. It is likely to be one amino acid larger than the G. sulfurreducens protein [17] which has a mass of 9.57 +0.02 kDa. The overall midpoint potential is measured at 791 mV, considerably more positive than the 7190 mV reported previously [16]. Our titration used a much wider range of redox potentials than used to study the cytochromes from either G. metallireducens [16] or G. sulfurreducens [17]. The non-sigmoidal shape of the redox titration indicates non-equivalent mid-point potentials for the haems, which is expected if the redox centers are not in identical amino acid environments [50]. This is true for the mid-point potentials of the haems in cytochrome c7 from D. acetoxidans, which were measured as 7102, 7177, and 7177 mV [51]. The yield of purified cytochrome, 0.1%, can be used to estimate the number of polypeptides in an average cell. Assuming a protein content similar to Escherichia coli of
1.5610715 g per cell [52], and a molecular weight of 9684, there are approximately 10 000 copies of this protein per cell. This would be ample cell associated protein to facilitate electron transport. Consideration of the amino acid sequence of the protein (Figure 1), including new sequence data, can be used to infer additional properties of the cytochrome. Previously described cytochrome c7 proteins have been localized to the periplasm [17]. The G. metallireducens c7 has an N-terminus of Ala-Asp indicative of a signal peptide cleavage site [53], and therefore supports the hypothesis that this is a periplasmic protein. The partial sequence data indicates no hydrophobic regions, so unlike the protein described in G. sulfurreducens [17], there is no evidence for membrane association. Also, that protein is described as having a near-neutral isoelectric point, while the sequence data for the G. metallireducens protein indicates a very basic protein (pI > 10). The basic nature of the G. metallireducens protein is further supported by its binding to carboxymethyl-cellulose, and the lysine-rich content [16]. It is unlikely that this positively charged hydrophilic protein would be membrane associated. Amino acid sequence data provides a molecular basis for the assignment of the G. metallireducens protein as a cytochrome c7. The haem 1 binding site of the D. acetoxidans protein is Cys-Asp-Ala-Cys-His (residues 26 to 30 of the mature D. acetoxidans protein sequence) and the additional His ligand is at amino acid 17 [31]. Residues 27 to 31, Cys-Lys-Lys-Cys-His in the G. metallireducens resembles the haem 1 binding site in the D. acetoxidans protein. Both have in common the His at position number 17, as well as a His at position number 20 which is the accessory ligand for haem 3 in the D. acetoxidans protein. Class III cytochromes have bis-histidinyl coordination, and therefore at least six histidines are expected. Afkar and Fukumori [16] report only four, while D. acetoxidans has six [54] and we estimate as many as ten in the G. metallireducens protein (data not shown). These similarities, and other amino acid identities suggest the gene that codes for the G. metallireducens protein is of the same gene family that codes for c7 and c3 cytochromes. Trihaem cytochromes c7 are homologous to the c3 cytochrome of Desulfovibrio without the haem 2 of the tetrahaem protein [31]. The presence of this cytochrome extends the recognized physiological similarities between the genera Geobacter and Desulfuromonas. These properties include an unusual, low-potential 2-oxoglutarate dehydrogenase [13] and the presence of menaquinone rather than ubiquinone [15]. Also, the documentation of these c7 cytochromes in iron-, humics-, and sulfurreducing anaerobes may help elucidate the mechanism of electron transport to these insoluble substrates.
ElectronTransfer in Geobacter metallireducens Acknowledgments This work was supported by a grant from the Office of Naval Research, Grant # N00014-91-J-1898, and a grant from the Grants and Research Funding Committee, Southeast Missouri State University. We would like to thank Ed Towers and Phil Ryan for technical assistance with the amino acid sequence analysis and Derek Lovley and John Stolz for useful discussion.
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