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Purification and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens
FEMS Microbiology Letters 175 (1999) 205^210
Puri¢cation and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens Eman Afkar a , Yoshihiro Fukumori b; * a
Department of Bioscience, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, Japan b Department of Biology, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan Received 22 March 1999; received in revised form 19 April 1999; accepted 19 April 1999
Abstract A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be 3190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7 , G. metallireducens cytochrome c did not show Fe(III)-reducing activity. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Geobacter metallireducens ; Cytochrome c; Iron respiration
1. Introduction Geobacter metallireducens, which is a strict anaerobic bacterium, was isolated from freshwater sediments of the Potomac River by Lovley and Phillips [1]. The bacterium can completely oxidize organic compounds by coupling with the reduction of Fe(III) as an electron acceptor. Therefore, it is generally believed that Fe(III) reduction and probably Mn(IV) reduction in sedimentary environments are primarily the result of the enzymic activity of specialized Fe(III)- and Mn(IV)-reducing microorgan* Corresponding author. Tel.: +81 (76) 2645719; Fax: +81 (76) 2645978; E-mail: [email protected]
isms, although little is known about the molecular mechanism of Fe(III) and Mn(IV) reduction in these bacteria [2]. Recently, Seeliger et al. have reported that the periplasmic cytochrome c with the molecular mass of 9.6 kDa may act as a Fe(III) reductase in G. sulfurreducens, which is a dissimilatory metal- and sulfur-reducing bacterium [3], while Gaspard et al. have reported that the iron reductase of G. sulfurreducens may be a high molecular mass cytochrome c that is weakly bound to the membrane [4]. In the present study, we ¢rst puri¢ed electrophoretically homogeneous cytochrome c7 , which is a sub-group of c-type cytochromes, from G. metallireducens and then investigated its molecular and structural features.
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 9 9 - 8
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2. Materials and methods
2.4. Measurement of Fe(III)-reducing activity
2.1. Bacterial growth
The iron reductase activity was assayed as previously described [4]. The reaction mixture, which contained 50 mM HEPES bu¡er (pH 7.0), 4 WM dithionite-reduced horse heart cytochrome c as an electron donor, 0.5 mM ferrozine as a Fe(II)-chelating agent, and 0.2 mM Fe(III) citrate as an electron acceptor, was placed in the main chamber of a Thunberg-type cuvette, and the intact cells, cell-free extract and membrane pellets (0.4 mg ml31 ) were placed in the side arm. Complexation of the reduced Fe(II) and ferrozine was followed by monitoring the absorbance at 562 nm with time and the reduction of the cytochrome was followed by measuring the increase in the absorbance at 550 nm. Iron-reducing activity of the puri¢ed cytochrome c was measured following the same method as described above.
G. metallireducens was kindly provided by Derek Lovley, Amherst, MA, USA. The bacterium was grown on aferric citrate medium [5]. In addition, 1% (v/v) each of vitamin solution and mineral solution were added [6]. The large-scale cultivation was performed in 9 l of the above medium in a 10-l bottle. The medium was inoculated with seed culture (1 l) under continuous £ushing with 80% N2 and 20% CO2 and autoclaved at 30³C for 3 days. The cells were harvested at the stationary phase, and about 60 g (wet weight) were obtained from 120 l of medium. 2.2. Physical and chemical measurements Spectrophotometric measurements were performed with a Shimadzu MPS 2000 spectrophotometer and 1 cm light path cuvette. The heme c content was determined spectrophotometrically by use of the millimolar extinction coe¤cient at 550 nm of pyridine ferrohemochrome c, 29.1 mM31 cm31 [7]. Polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate (SDS-PAGE) was performed according to the method of Schaëgger and Von Jagow [8]. The heme c in the gel was detected by heme c staining reagents [9]. The protein concentration was determined by a Bio-Rad DC reagent kit (catalog number 500-0116) with a slight modi¢cation proposed by Lowry et al., using bovine serum albumin as standard [10]. The amino acid composition of the puri¢ed cytochrome c was analyzed with amino acid analyzer (IRICA model g870 with autoreader g986, Kyoto, Japan) after hydrolysis with 6 N HCl at different time intervals (48 and 72 h) at 107³C. The N-terminal amino acid sequence of the puri¢ed cytochrome c was determined with a protein sequencer (Applied Biosystems, Model 478A). 2.3. Measurement of the redox potential We determined the midpoint redox potential using a spectroscopic titration method [11].
2.5. Metal content determination We determined the iron content of the puri¢ed protein using an atomic absorption/£ame emission spectrophotometry system (model AA-640-13). The standard curve was obtained with ferric nitrate solution. 2.6. Chemicals All chemicals analyzed or used as reagents were of the highest commercially available grade.
3. Results and discussion 3.1. Puri¢cation of cytochrome c from G. metallireducens All puri¢cation steps were conducted at 4³C under aerobic conditions. Frozen cells (about 60 g wet weight) from 120 l of culture medium were suspended in 10 mM Tris-HCl bu¡er (pH 8.0) containing 1 mM EDTA and 10 WM phenylmethylsulfonyl £uoride (PMSF) and disrupted with French pressure cell at 1000 kgf cm32 three times. Unbroken cells were removed by centrifugation at 12 100Ug for
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207
Fig. 1. A: SDS-PAGE of the G. metallireducens 9.5-kDa cytochrome c. The gel was stained with heme-staining reagents (lane 1) and Coomassie brilliant blue R-250 (lane 2). Marker proteins (lane 3) are carbonic anhydrase (31 000), soybean trypsin inhibitor doublet (20 400/ 19 700), horse heart myoglobin (16 900), lysozyme (14 400) and aprotinin (6100). B: Absorption spectra of the G. metallireducens 9.5-kDa cytochrome c. The protein was dissolved in 10 mM Tris-HCl bu¡er (pH 8.0), 1 mM EDTA, 10 WM PMSF and 0.1% Tween 20. Solid line: oxidized ; dashed line: reduced with dithionite. The inset shows the absorption spectrum of the pyridine ferrohemochrome of the G. metallireducens 9.5-kDa cytochrome c. To 17 WM cytochrome c were added 0.2 N NaOH, 10% pyridine, and a small amount of Na2 S2 O4 .
20 min. The resulting supernatant was subjected to ultracentrifugation at 185 000Ug for 2 h to remove the membrane pellets. The soluble fractions were charged on a Fractogel TSK DEAE 650-M column (2.5U16 cm), equilibrated with 10 mM Tris-HCl bu¡er (pH 8.0) containing 1 mM EDTA, 10 WM PMSF, and 0.1% Tween 20 (bu¡er A). The cytochrome c was eluted with a linear gradient produced è containfrom 300 ml each of bu¡er A and bu¡er A ing 0.5 M NaCl. The fraction containing cytochrome c was pooled, dialyzed against bu¡er A, and then charged on a Fractogel TSK DEAE 650 M column (2.5U10 cm) equilibrated with bu¡er A. The cytochrome c was eluted with a linear gradient of NaCl (0^0.5 M). The fraction containing cytochrome c was subjected to ammonium sulfate fractionation up to 55% saturation to remove the other respiratory components and dialysed against bu¡er A. After concentration with an Amicon ¢lter unit ¢tted with a cut o¡ membrane of 10 kDa (Microcon, Amicon, Beverly, MA, USA) the cytochrome c was loaded on the Sephacryl S-300 column (2U74 cm), equilibrated with bu¡er A containing 0.25 M NaCl, and eluted at a
Table 1 Amino acid composition of the 9.5-kDa cytochrome c of G. metallireducens Amino acid
Number of residuesa (per mol cytochrome c)
Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile Leu Phe Lys His Arg Total
10 5 2 10 4 10 6 7 7 1 3 4 4 18 4 1 96
a
The mol cytochrome c was calculated from the total proteins, assuming that the molecular mass of the cytochrome c is 9.5 kDa.
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ing reagents. These results revealed that the cytochrome c preparation was electrophoretically homogeneous. 3.2. Molecular features of the 9.5-kDa cytochrome c Fig. 2. Alignment of the N-terminal amino acid sequence of the G. metallireducens 9.5-kDa cytochrome c with those of D. acetoxidans cytochrome c7 and D. acetoxidans cytochrome c3 . The conserved amino acids are boxed.
£ow rate of 2.2 ml every 15 min. The fraction containing cytochrome c was pooled and used as the ¢nal preparation. About 1.28 mg of protein was obtained from 60 g wet cells. Fig. 1A shows the SDS-PAGE of the cytochrome c preparation obtained in the present study. One protein band with the molecular mass of 9.5 kDa was observed in the gel and stained with heme-stain-
The 9.5-kDa cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form as shown in Fig. 1B. The alkaline pyridine ferrohemochrome spectrum showed an absorption peak at 550 nm. Therefore, we concluded that the cytochrome contains heme c as a prosthetic group. The heme c content was estimated to be 231 nmol heme c mg protein31 on the basis of the OmM value of 29.1 mM31 of the pyridine ferrohemochrome of heme c. The metal content analysis showed that the 9.5-kDa cytochrome c contains 3 g atoms of iron per mol of cytochrome c. Furthermore, as presented in Table 1, the cytochrome c has seven cysteine residues in the
Fig. 3. Potentiometric titration of the puri¢ed 9.5-kDa G. metallireducens cytochrome c. To a Thunberg-type cuvette was added 1.3 WM of the puri¢ed cytochrome c which was suspended in 10 mM Tris-HCl bu¡er pH 7.0 and evacuated for 5 min. After complete reduction of the cytochrome c with dithionite, an anaerobic solution of 10 mM £avin mononucleotide (FMN) was added stepwise, and the decrease in the absorbance at 500 nm was followed. The midpoint redox potential of FMN was 3190 mV. The symbol b represents the observed values. The solid line was drawn according to the Nernst equation (n = 1).
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molecule. Therefore, we conclude that the 9.5-kDa cytochrome c is a multiheme protein. The N-terminal amino acid sequence of the ¢rst 17 residues of the 9.5-kDa cytochrome c was determined to be ADELTFKAKNGDVKFPP. Fig. 2 shows the alignment of the N-terminal amino acid sequence of the G. metallireducens 9.5-kDa cytochrome c with those of Desulfuromonas acetoxidans cytochrome c7 [12] and D. acetoxidans cytochrome c3 [13], suggesting that the G. metallireducens 9.5-kDa cytochrome c is very similar to the D. acetoxidans cytochrome c7 . Furthermore, the amino acid composition is also similar to that of the D. acetoxidans cytochrome c7 (data not shown). These results strongly suggest that the 9.5-kDa cytochrome c puri¢ed from G. metallireducens in the present study belongs to cytochrome c7 . The mid-point redox potential at pH 7.0 of the 9.5-kDa cytochrome c was determined to be 3190 mV (Fig. 3). The n-value of the Nernst equation was determined to be 1. Although the redox potential is very low, the ferrocytochrome c was not autoxidizable, suggesting that the coordinations of the hemes c were not modi¢ed under aerobic conditions. 3.3. Involvement of the 9.5-kDa cytochrome c in iron respiration of G. metallireducens Gaspard et al. reported that the iron reductase of G. sulfurreducens may be a high molecular mass cytochrome c which is weakly bound to the membrane [4]. Furthermore, Gorby and Lovley studied the electron transport of G. metallireducens and found that Fe(III) reductase is located in the membrane [14]. These results suggest that the G. metallireducens cytochrome c7 is not Fe(III) reductase. However, it should be noted that the triheme cytochrome c with a molecular mass of 9.6 kDa of G. sulfurreducens is excreted into the culture medium as Fe(III) reductase to donate electrons to insoluble iron hydroxides [3], and that D. acetoxidans cytochrome c7 shows Fe(III)-reducing activity [15]. In order to investigate the involvement of 9.5-kDa cytochrome c in the iron respiration of G. metallireducens, we compared the cytochrome c compositions of the soluble fraction prepared under anaerobic conditions from Fe(III)-grown cells or nitrate-grown cells. The total concentrations of heme c in the solu-
209
Fig. 4. SDS-PAGE of the soluble fractions prepared from Fe(III)-grown cells of G. metallireducens. The gel was stained with the heme-staining reagents.
ble fractions were determined to be about 151 WM and 13 WM, respectively. The soluble respiratory cytochromes c were expressed 12-fold more in Fe(III)grown cells than in nitrate-grown cells. Fig. 4 shows the gel with heme-staining reagents on SDS-PAGE of the soluble fraction prepared from Fe(III)-grown cells. Among the four kinds of cytochrome c, the 9.5kDa cytochrome c is highly expressed in the cells. However, neither the soluble fraction nor the puri¢ed cytochrome c7 showed Fe(III)-reducing activity. These results strongly suggest that although cytochrome c7 may participate in the anaerobic iron respiration of G. metallireducens, it is not Fe(III) reductase. The iron reductase activity of G. metallireducens could be observed with NADH as a physiological electron donor [16] and was inhibited by rotenone [14], which is a strong inhibitor of complex I. Therefore, further puri¢cation and biochemical characterization of the other respiratory components should be accomplished in the future to determine the molecular mechanism of iron reduction in the strict anaerobe G. metallireducens.
Acknowledgments We would like to thank Derek Lovley for providing Geobacter metallireducens. We would like to thank Tairo Oshima, Akihiko Yamagishi and Tomoko Yamazaki for their kind help in the anaerobic cultivation of the bacterium. This work was supported in part by a Grant-in-Aid for Scienti¢c Re-
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search (C) (09660076) and a Grant-in-Aid for Scienti¢c Research on Priority Areas (10129208) to Y.F. from the Ministry of Education, Science, Sports and Culture of Japan.
[9] [10]
References
[11]
[1] Lovley, D.R. and Phillips, E.J.P. (1986) Organic matter mineralization with the reduction of Ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51, 683^689. [2] Lovley, D.R. (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55, 259^287. [3] Seeliger, S., Ruwisch-Cord, R. and Bernhard, S. (1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as ferric iron reductase and as an electron carrier to other acceptors or to partner bacteria. J. Bacteriol. 180, 3686^3691. [4] Gaspard, S., Francisco, V. and Holliger, C. (1998) localization and solubilization of the iron(III) reductase of Geobacter sulfurreducens. Appl. Environ. Microbiol. 64, 3188^3194. [5] Lovley, D.R. and Phillips, E.J.P. (1988) Novel mode for microbial energy metabolism: organic matter oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54, 1472^1480. [6] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, R. and Wolfe, R.S. (1979) Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43, 260^269. [7] Yamanaka, T. (1988) Hemes. In: Metalloproteins, Chemical Properties and Biological E¡ects (Otsuka, S. and Yamanaka, T., Eds.), pp. 95^99. Kodansha, Tokyo. [8] Schaëgger, H. and Von Jagow, G. (1987) Tricine-sodium do-
[12]
[13]
[14]
[15]
[16]
decyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368^379. Connelly, J.L., Morrison, M. and Stots, E. (1958) Hemins of beef heart muscle. J. Biol. Chem. 233, 743^747. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265^275. Dutton, P.L. (1978) Redox potentiometry : determination of mid-point potentials of oxidation-reduction : reduction components of biological electron-transfer systems. Methods Enzymol. 54, 411^ 435. Banci, L., Bertini, I., Bruschi, M., Sompornpisut, P. and Turano, P. (1996) NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans. Proc. Natl. Acad. Sci. USA 93, 14396^ 14400. Bruschi, M., Woudstra, M., Guigliarelli, B., Asso, M., Lojou, E., Petillot, Y. and Abergel, C. (1997) Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans. Biochemistry 36, 10601^ 10608. Gorby, Y. and Lovley, D.R. (1991) Electron transport mechanism in dissimilatory Fe(III) reducing microorganism GS-15. Appl. Environ. Microbiol. 57, 867^870. Aubert, C., Lojou, E., Bianco, P., Rousset, M., Durand, M., Bruschi, M. and Dolla, A. (1998) The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity. Appl. Environ. Microbiol. 64, 1308^1312. Myres, C.R. and Myres, G.M. (1993) Ferric reductase was associated with the membrane of anaerobically grown Shewanella putrefaciens MR-I. FEMS Microbiol. Lett. 108, 15^ 22.
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Puri¢cation and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens Eman Afkar a , Yoshihiro Fukumori b; * a
Department of Bioscience, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, Japan b Department of Biology, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan Received 22 March 1999; received in revised form 19 April 1999; accepted 19 April 1999
Abstract A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be 3190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7 , G. metallireducens cytochrome c did not show Fe(III)-reducing activity. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Geobacter metallireducens ; Cytochrome c; Iron respiration
1. Introduction Geobacter metallireducens, which is a strict anaerobic bacterium, was isolated from freshwater sediments of the Potomac River by Lovley and Phillips [1]. The bacterium can completely oxidize organic compounds by coupling with the reduction of Fe(III) as an electron acceptor. Therefore, it is generally believed that Fe(III) reduction and probably Mn(IV) reduction in sedimentary environments are primarily the result of the enzymic activity of specialized Fe(III)- and Mn(IV)-reducing microorgan* Corresponding author. Tel.: +81 (76) 2645719; Fax: +81 (76) 2645978; E-mail: [email protected]
isms, although little is known about the molecular mechanism of Fe(III) and Mn(IV) reduction in these bacteria [2]. Recently, Seeliger et al. have reported that the periplasmic cytochrome c with the molecular mass of 9.6 kDa may act as a Fe(III) reductase in G. sulfurreducens, which is a dissimilatory metal- and sulfur-reducing bacterium [3], while Gaspard et al. have reported that the iron reductase of G. sulfurreducens may be a high molecular mass cytochrome c that is weakly bound to the membrane [4]. In the present study, we ¢rst puri¢ed electrophoretically homogeneous cytochrome c7 , which is a sub-group of c-type cytochromes, from G. metallireducens and then investigated its molecular and structural features.
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 9 9 - 8
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2. Materials and methods
2.4. Measurement of Fe(III)-reducing activity
2.1. Bacterial growth
The iron reductase activity was assayed as previously described [4]. The reaction mixture, which contained 50 mM HEPES bu¡er (pH 7.0), 4 WM dithionite-reduced horse heart cytochrome c as an electron donor, 0.5 mM ferrozine as a Fe(II)-chelating agent, and 0.2 mM Fe(III) citrate as an electron acceptor, was placed in the main chamber of a Thunberg-type cuvette, and the intact cells, cell-free extract and membrane pellets (0.4 mg ml31 ) were placed in the side arm. Complexation of the reduced Fe(II) and ferrozine was followed by monitoring the absorbance at 562 nm with time and the reduction of the cytochrome was followed by measuring the increase in the absorbance at 550 nm. Iron-reducing activity of the puri¢ed cytochrome c was measured following the same method as described above.
G. metallireducens was kindly provided by Derek Lovley, Amherst, MA, USA. The bacterium was grown on aferric citrate medium [5]. In addition, 1% (v/v) each of vitamin solution and mineral solution were added [6]. The large-scale cultivation was performed in 9 l of the above medium in a 10-l bottle. The medium was inoculated with seed culture (1 l) under continuous £ushing with 80% N2 and 20% CO2 and autoclaved at 30³C for 3 days. The cells were harvested at the stationary phase, and about 60 g (wet weight) were obtained from 120 l of medium. 2.2. Physical and chemical measurements Spectrophotometric measurements were performed with a Shimadzu MPS 2000 spectrophotometer and 1 cm light path cuvette. The heme c content was determined spectrophotometrically by use of the millimolar extinction coe¤cient at 550 nm of pyridine ferrohemochrome c, 29.1 mM31 cm31 [7]. Polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate (SDS-PAGE) was performed according to the method of Schaëgger and Von Jagow [8]. The heme c in the gel was detected by heme c staining reagents [9]. The protein concentration was determined by a Bio-Rad DC reagent kit (catalog number 500-0116) with a slight modi¢cation proposed by Lowry et al., using bovine serum albumin as standard [10]. The amino acid composition of the puri¢ed cytochrome c was analyzed with amino acid analyzer (IRICA model g870 with autoreader g986, Kyoto, Japan) after hydrolysis with 6 N HCl at different time intervals (48 and 72 h) at 107³C. The N-terminal amino acid sequence of the puri¢ed cytochrome c was determined with a protein sequencer (Applied Biosystems, Model 478A). 2.3. Measurement of the redox potential We determined the midpoint redox potential using a spectroscopic titration method [11].
2.5. Metal content determination We determined the iron content of the puri¢ed protein using an atomic absorption/£ame emission spectrophotometry system (model AA-640-13). The standard curve was obtained with ferric nitrate solution. 2.6. Chemicals All chemicals analyzed or used as reagents were of the highest commercially available grade.
3. Results and discussion 3.1. Puri¢cation of cytochrome c from G. metallireducens All puri¢cation steps were conducted at 4³C under aerobic conditions. Frozen cells (about 60 g wet weight) from 120 l of culture medium were suspended in 10 mM Tris-HCl bu¡er (pH 8.0) containing 1 mM EDTA and 10 WM phenylmethylsulfonyl £uoride (PMSF) and disrupted with French pressure cell at 1000 kgf cm32 three times. Unbroken cells were removed by centrifugation at 12 100Ug for
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Fig. 1. A: SDS-PAGE of the G. metallireducens 9.5-kDa cytochrome c. The gel was stained with heme-staining reagents (lane 1) and Coomassie brilliant blue R-250 (lane 2). Marker proteins (lane 3) are carbonic anhydrase (31 000), soybean trypsin inhibitor doublet (20 400/ 19 700), horse heart myoglobin (16 900), lysozyme (14 400) and aprotinin (6100). B: Absorption spectra of the G. metallireducens 9.5-kDa cytochrome c. The protein was dissolved in 10 mM Tris-HCl bu¡er (pH 8.0), 1 mM EDTA, 10 WM PMSF and 0.1% Tween 20. Solid line: oxidized ; dashed line: reduced with dithionite. The inset shows the absorption spectrum of the pyridine ferrohemochrome of the G. metallireducens 9.5-kDa cytochrome c. To 17 WM cytochrome c were added 0.2 N NaOH, 10% pyridine, and a small amount of Na2 S2 O4 .
20 min. The resulting supernatant was subjected to ultracentrifugation at 185 000Ug for 2 h to remove the membrane pellets. The soluble fractions were charged on a Fractogel TSK DEAE 650-M column (2.5U16 cm), equilibrated with 10 mM Tris-HCl bu¡er (pH 8.0) containing 1 mM EDTA, 10 WM PMSF, and 0.1% Tween 20 (bu¡er A). The cytochrome c was eluted with a linear gradient produced è containfrom 300 ml each of bu¡er A and bu¡er A ing 0.5 M NaCl. The fraction containing cytochrome c was pooled, dialyzed against bu¡er A, and then charged on a Fractogel TSK DEAE 650 M column (2.5U10 cm) equilibrated with bu¡er A. The cytochrome c was eluted with a linear gradient of NaCl (0^0.5 M). The fraction containing cytochrome c was subjected to ammonium sulfate fractionation up to 55% saturation to remove the other respiratory components and dialysed against bu¡er A. After concentration with an Amicon ¢lter unit ¢tted with a cut o¡ membrane of 10 kDa (Microcon, Amicon, Beverly, MA, USA) the cytochrome c was loaded on the Sephacryl S-300 column (2U74 cm), equilibrated with bu¡er A containing 0.25 M NaCl, and eluted at a
Table 1 Amino acid composition of the 9.5-kDa cytochrome c of G. metallireducens Amino acid
Number of residuesa (per mol cytochrome c)
Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile Leu Phe Lys His Arg Total
10 5 2 10 4 10 6 7 7 1 3 4 4 18 4 1 96
a
The mol cytochrome c was calculated from the total proteins, assuming that the molecular mass of the cytochrome c is 9.5 kDa.
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ing reagents. These results revealed that the cytochrome c preparation was electrophoretically homogeneous. 3.2. Molecular features of the 9.5-kDa cytochrome c Fig. 2. Alignment of the N-terminal amino acid sequence of the G. metallireducens 9.5-kDa cytochrome c with those of D. acetoxidans cytochrome c7 and D. acetoxidans cytochrome c3 . The conserved amino acids are boxed.
£ow rate of 2.2 ml every 15 min. The fraction containing cytochrome c was pooled and used as the ¢nal preparation. About 1.28 mg of protein was obtained from 60 g wet cells. Fig. 1A shows the SDS-PAGE of the cytochrome c preparation obtained in the present study. One protein band with the molecular mass of 9.5 kDa was observed in the gel and stained with heme-stain-
The 9.5-kDa cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form as shown in Fig. 1B. The alkaline pyridine ferrohemochrome spectrum showed an absorption peak at 550 nm. Therefore, we concluded that the cytochrome contains heme c as a prosthetic group. The heme c content was estimated to be 231 nmol heme c mg protein31 on the basis of the OmM value of 29.1 mM31 of the pyridine ferrohemochrome of heme c. The metal content analysis showed that the 9.5-kDa cytochrome c contains 3 g atoms of iron per mol of cytochrome c. Furthermore, as presented in Table 1, the cytochrome c has seven cysteine residues in the
Fig. 3. Potentiometric titration of the puri¢ed 9.5-kDa G. metallireducens cytochrome c. To a Thunberg-type cuvette was added 1.3 WM of the puri¢ed cytochrome c which was suspended in 10 mM Tris-HCl bu¡er pH 7.0 and evacuated for 5 min. After complete reduction of the cytochrome c with dithionite, an anaerobic solution of 10 mM £avin mononucleotide (FMN) was added stepwise, and the decrease in the absorbance at 500 nm was followed. The midpoint redox potential of FMN was 3190 mV. The symbol b represents the observed values. The solid line was drawn according to the Nernst equation (n = 1).
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molecule. Therefore, we conclude that the 9.5-kDa cytochrome c is a multiheme protein. The N-terminal amino acid sequence of the ¢rst 17 residues of the 9.5-kDa cytochrome c was determined to be ADELTFKAKNGDVKFPP. Fig. 2 shows the alignment of the N-terminal amino acid sequence of the G. metallireducens 9.5-kDa cytochrome c with those of Desulfuromonas acetoxidans cytochrome c7 [12] and D. acetoxidans cytochrome c3 [13], suggesting that the G. metallireducens 9.5-kDa cytochrome c is very similar to the D. acetoxidans cytochrome c7 . Furthermore, the amino acid composition is also similar to that of the D. acetoxidans cytochrome c7 (data not shown). These results strongly suggest that the 9.5-kDa cytochrome c puri¢ed from G. metallireducens in the present study belongs to cytochrome c7 . The mid-point redox potential at pH 7.0 of the 9.5-kDa cytochrome c was determined to be 3190 mV (Fig. 3). The n-value of the Nernst equation was determined to be 1. Although the redox potential is very low, the ferrocytochrome c was not autoxidizable, suggesting that the coordinations of the hemes c were not modi¢ed under aerobic conditions. 3.3. Involvement of the 9.5-kDa cytochrome c in iron respiration of G. metallireducens Gaspard et al. reported that the iron reductase of G. sulfurreducens may be a high molecular mass cytochrome c which is weakly bound to the membrane [4]. Furthermore, Gorby and Lovley studied the electron transport of G. metallireducens and found that Fe(III) reductase is located in the membrane [14]. These results suggest that the G. metallireducens cytochrome c7 is not Fe(III) reductase. However, it should be noted that the triheme cytochrome c with a molecular mass of 9.6 kDa of G. sulfurreducens is excreted into the culture medium as Fe(III) reductase to donate electrons to insoluble iron hydroxides [3], and that D. acetoxidans cytochrome c7 shows Fe(III)-reducing activity [15]. In order to investigate the involvement of 9.5-kDa cytochrome c in the iron respiration of G. metallireducens, we compared the cytochrome c compositions of the soluble fraction prepared under anaerobic conditions from Fe(III)-grown cells or nitrate-grown cells. The total concentrations of heme c in the solu-
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Fig. 4. SDS-PAGE of the soluble fractions prepared from Fe(III)-grown cells of G. metallireducens. The gel was stained with the heme-staining reagents.
ble fractions were determined to be about 151 WM and 13 WM, respectively. The soluble respiratory cytochromes c were expressed 12-fold more in Fe(III)grown cells than in nitrate-grown cells. Fig. 4 shows the gel with heme-staining reagents on SDS-PAGE of the soluble fraction prepared from Fe(III)-grown cells. Among the four kinds of cytochrome c, the 9.5kDa cytochrome c is highly expressed in the cells. However, neither the soluble fraction nor the puri¢ed cytochrome c7 showed Fe(III)-reducing activity. These results strongly suggest that although cytochrome c7 may participate in the anaerobic iron respiration of G. metallireducens, it is not Fe(III) reductase. The iron reductase activity of G. metallireducens could be observed with NADH as a physiological electron donor [16] and was inhibited by rotenone [14], which is a strong inhibitor of complex I. Therefore, further puri¢cation and biochemical characterization of the other respiratory components should be accomplished in the future to determine the molecular mechanism of iron reduction in the strict anaerobe G. metallireducens.
Acknowledgments We would like to thank Derek Lovley for providing Geobacter metallireducens. We would like to thank Tairo Oshima, Akihiko Yamagishi and Tomoko Yamazaki for their kind help in the anaerobic cultivation of the bacterium. This work was supported in part by a Grant-in-Aid for Scienti¢c Re-
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search (C) (09660076) and a Grant-in-Aid for Scienti¢c Research on Priority Areas (10129208) to Y.F. from the Ministry of Education, Science, Sports and Culture of Japan.
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