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The redox centers in the molybdo iron-sulfur flavoprotein CO dehydrogenase from the thermophilic carboxidotrophic bacterium Pseudomonas thermocarboxydovorans
FEMS Microbiology Letters 176 (1999) 139^145
The redox centers in the molybdo iron-sulfur £avoprotein CO dehydrogenase from the thermophilic carboxidotrophic bacterium Pseudomonas thermocarboxydovorans Petra Ha«nzelmann, Bettina Hofmann, Sabine Meisen, Ortwin Meyer * Lehrstuhl fu«r Mikrobiologie, Universita«t Bayreuth, Universita«tsstraMe 30, D-95440 Bayreuth, Germany Received 5 March 1999; received in revised form 4 May 1999; accepted 4 May 1999
Abstract The redox centers in the molybdo iron-sulfur flavoprotein CO dehydrogenase from the thermophilic bacterium Pseudomonas thermocarboxydovorans were identified and characterized by electron paramagnetic resonance (EPR). One mol of the 279-kDa dimer contained 1.9 mol of Mo, 2.2 mol of FAD, 6.9 mol of Fe and 6.7 mol of labile sulfide. The molybdenum cofactor is composed of a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and the Mo ion. EPR spectroscopy revealed signals typical for Mo(V), FADH , and type I and type II [2Fe-2S] centers. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Carbon monoxide ; Carboxidotrophic bacterium; CO dehydrogenase; Molybdenum cofactor; Molybdopterin-cytosine dinucleotide
1. Introduction The CO dehydrogenases of the two mesophilic bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudo£ava are molybdo iron-sulfur £avoproteins [1^4]. The enzyme from O. carboxidovorans is a 273.4-kDa dimer of LMS heterotrimers [1,2,5] î and its crystal structure has been resolved at 2.2 A [6]. The L subunit is the molybdoprotein and contains a 1:1 mononuclear complex of a Mo ion and molybdopterin-cytosine dinucleotide (MCD). The M subunit is the £avoprotein and carries the FAD. The
* Corresponding author. Tel.: +49 (921) 552729; Fax: +49 (921) 552727; E-mail: [email protected]
S subunit is the iron-sulfur protein and carries a type I and a type II [2Fe-2S] center. The CO dehydrogenase from the thermophile Pseudomonas thermocarboxydovorans has the same subunit composition [7,8] as the enzyme from the mesophile O. carboxidovorans [1,2,5]. The CO dehydrogenases are members of the sequence family of molybdenum hydroxylases [9]. The enzyme subunits L, M and S of O. carboxidovorans and the subunits A, B and C of P. thermocarboxydovorans show signi¢cant sequence homologies [5,8]. The enzyme from P. thermocarboxydovorans has been identi¢ed as an iron-sulfur £avoprotein containing 2 mol of £avin and nearly 8 mol of Fe and 8 mol of acid labile sulfur per mol of enzyme [7]. The £avin and the iron-sulfur centers have not been analyzed. Although 0.7 mol
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 2 3 0 - X
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of Mo per mol of CO dehydrogenase has been demonstrated, no attempts have been made to study the Mo center by electron paramagnetic resonance (EPR) spectroscopy. The molybdenum cofactor of the CO dehydrogenase from P. thermocarboxydovorans has not been isolated and the type of pterin engaged in the coordinative binding of the Mo ion is not known. That the pterin will be MCD [8] has been assumed from the presence of MCD in the molybdenum cofactor of the CO dehydrogenases from di¡erent bacteria [3], as well as from the similarities between the sequence 712L GGGFGNK718L on the large polypeptide (CutA) of the CO dehydrogenase from P. thermocarboxydovorans and the consensus sequence GXGX2 G for dinucleotide binding [10]. However, the sequences of pterin contacting or conserved segments, e.g. identi¢ed in the structurally characterized Mop protein from Desulfovibrio gigas [11], alone do not permit a solid conclusion on the type of pterin present in a protein. In this paper we report on the identi¢cation and characterization of the redox centers in CO dehydrogenase from P. thermocarboxydovorans.
drophobic interaction chromatography on Source 15 ISO (Pharmacia) and dye ligand a¤nity chromatography on Dyematrex red A (Millipore). The oxidation of CO by CO dehydrogenases was assayed spectrophotometrically [14]. One unit of CO dehydrogenase activity is de¢ned as the amount catalyzing the reduction of 1 Wmol 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride min31 at 30³C.
2. Materials and methods
2.4. Miscellaneous methods
2.1. Bacterial strains and cultivation
Analytical PAGE was carried out on a discontinuous system [17] employing a 5% stacking gel and a 7.5% running gel (native PAGE) or a 7.5% stacking gel and a 12% running gel (SDS-PAGE). Gels were stained for protein with Coomassie brilliant blue G250. The N-termini of subunits were sequenced as described [18]. The method of Bradford [19] was employed for the estimation of soluble proteins. Homogeneous CO dehydrogenases were also quantitated by their absorption at 450 nm employing a millimolar extinction coe¤cient at 450 nm of 72 mM31 cm31 and molecular masses of 283.121 kDa (P. thermocarboxydovorans [8]) or 277.436 kDa (O. carboxidovorans [5]). Analyses of Fe [20], Mo [21] and acid labile sulfur [22] followed published procedures. X-band EPR spectra were recorded on a Bruker EMX spectrometer equipped with a ESR 900 helium cryostat (Oxford Instruments) under the experimental conditions described [4,23].
Bacterial cell mass was produced with CO as the substrate under chemolithoautotrophic conditions in 70-l fermenters ¢lled with 50 l of a mineral medium of pH 7.2 plus trace element solution TS2 containing ammonium Fe(III) citrate (0.1 g l31 ) and ammonium chloride (5 g l31 ) [12]. O. carboxidovorans OM5 (DSM 1227) was grown at 30³C [12] and P. thermocarboxydovorans strain C2 (NCIB 11893) at 50³C [13]. 2.2. Puri¢cation and assay of CO dehydrogenase Puri¢cation of the CO dehydrogenases from P. thermocarboxydovorans [7] or O. carboxidovorans [4] basically followed published protocols and involved precipitation of nucleic acids with 3 mg of protamine sulfate per g of bacterial cell mass, anion exchange chromatography on Source 30 Q (Pharmacia), hy-
2.3. Analysis of pterins, nucleotides and £avins Molybdopterin (MPT) dinucleotides in CO dehydrogenases were analyzed by oxidation with I2 /KI [15]. Carboxamidomethylation of pterins was with iodoacetamide after extraction from CO dehydrogenase with SDS [16] followed by isocratic reversed phase HPLC [4]. FAD, extracted from CO dehydrogenase with SDS, was analyzed by HPLC [4]. FAD was also analyzed spectrophotometrically in supernatants of TCA precipitates [1]. Nucleotides were released from MCD or FAD by hydrolysis of CO dehydrogenase with sulfuric acid followed by HPLC analysis [4].
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3. Results and discussion 3.1. Puri¢cation of CO dehydrogenase and some properties CO dehydrogenase was puri¢ed from P. thermocarboxydovorans about 9-fold with a yield of 22% and a speci¢c activity of 3.1 U mg protein31 . The homogeneity of the preparations is apparent from a single band upon native PAGE (Fig. 1A, lane 2) and the appearance of only the three enzyme polypeptides upon SDS-PAGE (Fig. 1B, lane 4). The mobilities of the CO dehydrogenases from P. thermocarboxydovorans and O. carboxidovorans on native PAGE (Fig. 1A) were in accordance with the molecular masses of (kDa) 279.174 [8] or 273.532 [5], respectively, calculated from the subunit amino acid composition. SDS-PAGE shows that the CO dehydrogenases from both sources have the same (LMS)2 subunit structure (Fig. 1B).
Fig. 2. Comparison of the UV/Vis absorption spectra of air-oxidized CO dehydrogenases (1.33 WM, in 50 mM Hepes, pH 7.2). Sources: P. thermocarboxydovorans, solid line; O. carboxidovorans, dotted line.
The N-termini of the P. thermocarboxydovorans CO dehydrogenase A subunit (NAPLSDREKA), B subunit (MIPPAFAYHA) and C subunit (SKHIVSMTVN) were identical to the sequences reported of CutA, CutB and CutC, respectively [8]. The enzyme has a typical CO dehydrogenase UV/ Vis absorption spectrum exhibiting a protein and cofactor absorption peak at 276 nm, FAD absorption maxima at 386 nm and around 450 nm plus absorptions of the [2Fe-2S] centers around 325, 420, 465 and 550 nm (Fig. 2, solid line). 3.2. Identi¢cation of a mononuclear 1:1 Mo-MCD complex
Fig. 1. Homogeneity and polypeptides of CO dehydrogenase. The CO dehydrogenases puri¢ed from O. carboxidovorans (lanes 1 and 3) or P. thermocarboxydovorans (lanes 2 and 4) were subjected to native (A) and SDS-PAGE (B). Native gels, 50 Wg protein; SDS gels, 70 Wg protein.
CO dehydrogenase from P. thermocarboxydovorans contained 1.94 þ 0.08 mol of Mo mol enzyme31 (three determinations). Iodine oxidation of the enzyme produced a compound with a £uorescence excitation maximum at 380 nm and an emission maximum at 460 nm indicative of form A MPT [24]. The relative £uorescence yields of form A MPT from CO dehydrogenase of P. thermocarboxydovorans (89.1 þ 14.6%) or O. carboxidovorans (set at 100%) indicate that both enzymes contain the same amounts of MPT. HPLC analysis of CO dehydrogenase treated with sulfuric acid revealed 1.91 þ 0.10 mol of 5P-CMP mol enzyme31 (three determinations), which was identi¢ed on the basis of co-elution
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with authentic 5P-CMP at 7.4 min (Fig. 3A). The contents of Mo, 5P-CMP and MPT indicate a mononuclear 1:1 Mo-MCD complex in the CO dehydrogenase from P. thermocarboxydovorans. Direct proof for the presence of MCD was obtained from alkylation of the enzyme with iodoacetamide under reducing conditions. Upon HPLC the eluting compound at 10.1 min was identi¢ed as di(carboxamidomethyl)molybdopterin-cytosine dinucleotide [di(cam)MCD] on the basis of its characteristic UV/Vis absorption spectrum with maxima at 278 nm and 367 nm, an OD278 /OD367 ratio of 3.3 and co-elution with authentic di(cam)MCD (Fig. 3B). 3.3. Characterization of the metal centers by EPR EPR spectroscopy of dithionite-reduced CO dehydrogenase from P. thermocarboxydovorans revealed at 120 K a rhombic Mo(V) signal with g factors of g1 = 1.978, g2 = 1.967 and g3 = 1.954 (gav = 1.966) and a line width of 7 mT (Fig. 4A, a). In addition to the intense signal of the 96 Mo isotope (74.5% natural abundance) without a nuclear spin, weaker signals arising from hyper¢ne interaction with the 95 Mo and 97 Mo nuclei, both with a nuclear spin of I = 5/ 2 and with natural abundances of 15.9% and 9.6%, are visible (Fig. 4A, a). Very similar Mo(V) signals have been reported for the CO dehydrogenases from O. carboxidovorans [23] and H. pseudo£ava [4]. Airoxidized CO dehydrogenase was EPR silent at 120 K (Fig. 4A, b), referring to a Mo(VI). Upon reduction with CO, CO dehydrogenase revealed at 120 K an isotropic signal (g = 2.005), typical of an organic radical, and additional periodically repeating signals (Fig. 4A, c). At 1 mW microwave power the signal at g = 2.005 has a peak to peak line width of 0.21 mT, indicative of the neutral FADH semiquinone radical [25]. The FADH radical has a maximum signal intensity at about 120 K and is saturated at P1=2 = 0.5 mW (Fig. 5). The additional periodically repeating EPR lines were saturated at P1=2 = 20 mW (Fig. 5). These signals showed maximum intensities below 75 K; the exact temperature could not be determined because below 70 K the [2Fe-2S] EPR signals became apparent. The crystal structure of CO dehydrogenase from O. carboxidovorans shows a catalytic essential selenium atom in a distance of î to the Mo ion [6]. The selenium is bound to 3.7 A
Fig. 3. A: HPLC elution of the 5P-nucleotides obtained through acid hydrolysis of the CO dehydrogenases from P. thermocarboxydovorans (1.71 nmol, trace I) or O. carboxidovorans (0.85 nmol, trace II) and the corresponding spectra of the materials eluting at 7.4 min and 17.4 min of trace I (inset). B: HPLC elution of the alkylated pterins isolated from the CO dehydrogenases from P. thermocarboxydovorans (14 nmol, trace I) or O. carboxidovorans (7 nmol, trace II) and the spectra of the materials eluting at 10.1 min (inset). C: HPLC elution of the £avins extracted with SDS from the CO dehydrogenases from P. thermocarboxydovorans (3.74 nmol, trace I) or O. carboxidovorans (1.87 nmol, trace II) and the spectra of the materials eluting at 22.8 min (inset).
the cysteine388L residue which is situated in a loop (384L VAYRCSFR391L ) in the active site. The P. thermocarboxydovorans CO dehydrogenase has the same sequence (378A VAYRCSFR385A ), including cysteine382A on CutA. Therefore, we ascribe the repeating EPR lines in the spectrum of Fig. 4A, c to a Mo/ Se center, produced upon substrate binding/oxidation.
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Fig. 4. Mo(V) and iron-sulfur EPR spectra of CO dehydrogenase from P. thermocarboxydovorans (35 WM, in 50 mM Hepes, pH 7.2). Reduction under anoxic conditions was with 9.5 mM dithionite or CO. Instrument settings : microwave frequency, 9.47 GHz; modulation amplitude, 1 mT. A: Spectra originating from Mo(V) were recorded at 120 K with a microwave power of 10 mW. a, dithionite-reduced ; the parts of the spectra at high and low g factors are shown with the vertical scale increased 5-fold ; b, air-oxidized ; c, CO-reduced. B: Iron-sulfur EPR was at 16 K with a microwave power of 200 mW or at 49 K with a microwave power of 10 mW. d, 16 K, dithionitereduced ; e, 16 K, air-oxidized ; f, 16 K, CO-reduced ; g, 49 K, dithionite-reduced ; h, 49 K, air-oxidized ; i, 49 K, CO-reduced. The numbers in the spectra indicate the g values.
P. thermocarboxydovorans CO dehydrogenase contained 6.90 þ 0.18 mol of Fe and 6.72 þ 0.18 mol of acid labile sulfur mol enzyme31 (three determinations). Dithionite-reduced CO dehydrogenase from P. thermocarboxydovorans showed at temperatures below 60 K rhombic EPR signals of two di¡erent [2Fe-2S] centers (Fig. 4B, d, g). As de¢ned with xanthine oxidase, and according to the convention in the literature [25], the rhombic [2Fe-2S] signal with the smaller g anisotropy is termed type I [g1 = 2.024, g2 = 1.945, g3 = 1.910, gav = 1.960 (Fig. 4B, g)], and that with the larger g anisotropy is termed type II [g1 = 2.158, g2 = 1.978, g3 = 1.895, gav = 2.010 (Fig. 4B, d)]. At 49 K almost only the signals of the type I center were detectable (Fig. 4B, g). At 16 K a combined EPR spectrum of the signals originating from the type I and the type II centers was visible (Fig. 4B, d). In addition, features due to the presence of Mo(V) at 49 K (g = 1.977 and g = 1.970) were present (Fig. 4B, g). The air-oxidized enzyme was EPR silent (Fig. 4B, e, h). The CO-reduced enzyme revealed at 16 K and 49 K signals of the type I and type II [2Fe-2S] centers (Fig. 4B, f, i).
Fig. 5. Microwave power saturation behavior of CO-reduced FADH and Mo(V) signals at 120 K. Spectra were recorded using the conditions given in Fig. 4A, except that the microwave power (P) was varied over the range of 0.01^200 mW. The signal intensities (S) of the following spectral features were measured : for FADH , the overall signal amplitude above and below baseline; for Mo(V), the overall signal amplitude of g1:946 above and below baseline. The determination of P1=2 was as described [23].
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3.4. Presence of FAD Trichloroacetic acid supernatants of CO dehydrogenase from P. thermocarboxydovorans revealed 2.20 þ 0.12 mol of £avin mol enzyme31 (three determinations), which was identi¢ed as FAD on the basis of the appearance of 1.76 þ 0.25 mol of 5P-AMP mol enzyme31 (three determinations) at 17.4 min upon HPLC of acid hydrolysates of CO dehydrogenase (Fig. 3A). HPLC analysis of CO dehydrogenase unfolded with SDS revealed a single compound absorbing at 450 nm which was identi¢ed as FAD on the basis of its characteristic UV/Vis absorption spectrum and co-elution with authentic FAD (Fig. 4C). The presence of FAD in CutB of CO dehydrogenase from P. thermocarboxydovorans is apparent from the sequences 32B AGGHS36B and 111B TIGG114B which have been identi¢ed in the high resolution structure of the CO dehydrogenase from O. carboxidovorans as £avin-binding [6].
[5]
[6]
[7]
[8]
[9] [10]
[11]
Acknowledgements We thank Dr. Heinz Faulhammer (Universita«t Bayreuth) for the determination of N-terminal amino acid sequences. This work was ¢nancially supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt am Main, Germany).
[12]
[13]
[14]
References [15] [1] Meyer, O. (1982) Chemical and spectral properties of carbon monoxide:methylene blue oxidoreductase. J. Biol. Chem. 257, 1333^1341. [2] Meyer, O., Frunzke, K. and Mo«rsdorf, G. (1993) Biochemistry of the aerobic utilization of carbon monoxide. In: Microbial Growth on C1 Compounds (Murrell, J.C. and Kelly, D.P., Eds.), pp. 433^459. Intercept Scienti¢c, Andover. [3] Meyer, O., Frunzke, K., Tachil, J. and Volk, M. (1993) The bacterial molybdenum cofactor. In: Molybdenum Enzymes, Cofactors, and Model Systems (Stiefel, E.I., Coucouvanis, D. and Newton, W.E., Eds.), pp. 50^68. ACS Symposium Series 535, American Chemical Society, Washington, DC. [4] Ha«nzelmann, P. and Meyer, O. (1998) E¡ect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine dinucleotide-type of molybdenum
[16]
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[19]
cofactor in Hydrogenophaga pseudo£ava. Eur. J. Biochem. 255, 755^765. Schu«bel, U., Kraut, M., Mo«rsdorf, G. and Meyer, O. (1995) Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans. J. Bacteriol. 177, 2197^2203. Dobbek, H., Gremer, L., Meyer, O. and Huber, R. (1999) Crystal structure of CO dehydrogenase, a molybdo iron-sulfur £avoprotein containing S-selanylcysteine. Nature, submitted. Bell, J.M., Williams, E. and Colby, J. (1985) Carbon monoxide oxidoreductase from thermophilic carboxydobacteria. In: Microbial Gas Metabolism (Poole, R.K. and Dow, C.S., Eds.), pp. 153^160. Academic Press, London. Pearson, D.M., O'Reilly, C., Colby, J. and Black, G.W. (1994) DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2. Biochim. Biophys. Acta 1188, 432^438. Hille, R. (1996) Structure and function of mononuclear molybdenum enzymes. J. Biol. Inorg. Chem. 1, 397^404. Wierenga, R.K., De Maeyer, M.C.H. and Hol, W.G.J. (1985) Interaction of pyrophosphate moieties with K-helices in dinucleotide binding proteins. Biochemistry 24, 1346^1357. Roma¬o, M.J., Archer, M., Moura, I., Moura, J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. and Huber, R. (1995) Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D. gigas. Science 270, 1170^1176. Meyer, O. and Schlegel, H.G. (1983) Biology of aerobic carbon monoxide-oxidizing bacteria. Annu. Rev. Microbiol. 37, 277^310. Lyons, C.M., Justin, P., Colby, J. and Williams, E. (1984) Isolation, characterization and autotrophic metabolism of a moderately thermophilic carboxydobacterium, Pseudomonas thermocarboxydovorans sp. nov.. J. Gen. Microbiol. 130, 1097^1105. Kraut, M., Hugendieck, I., Herwig, S. and Meyer, O. (1989) Homology and distribution of CO dehydrogenase structural genes in carboxydotrophic bacteria. Arch. Microbiol. 152, 335^341. Johnson, J.L. and Rajagopalan, K.V. (1982) Structural and metabolic relationship between the molybdenum cofactor and urothione. Proc. Natl. Acad. Sci. USA 79, 6856^6860. Johnson, J.L., Bastian, N.R. and Rajagopalan, K.V. (1990) Molybdopterin guanine dinucleotide: A modi¢ed form of molybdopterin identi¢ed in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitri¢cans. Proc. Natl. Acad. Sci. USA 87, 3190^3194. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. Gremer, L. and Meyer, O. (1996) Characterization of xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica and identi¢cation of a molybdopterin-cytosine-dinucleotide-containing molybdenum cofactor. Eur. J. Biochem. 238, 862^866. Bradford, M.M. (1976) A rapid and sensitive method for the
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P. Ha«nzelmann et al. / FEMS Microbiology Letters 176 (1999) 139^145 quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248^ 254. [20] Fish, W.W. (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol. 158, 357^364. [21] Cardenas, J. and Mortenson, L.E. (1974) Determination of molybdenum and tungsten in biological materials. Anal. Biochem. 60, 372^381. [22] Fogo, J.K. and Popowsky, M. (1949) Spectrophotometric determination of hydrogen sul¢de. Anal. Chem. 21, 732^734.
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[23] Bray, R.C., George, G.N., Lange, R. and Meyer, O. (1983) Studies by e.p.r. spectroscopy of carbon monoxide oxidases from Pseudomonas carboxydovorans and Pseudomonas carboxydohydrogena. Biochem. J. 211, 687^694. [24] Meyer, O. and Rajagopalan, K.V. (1984) Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. J. Bacteriol. 157, 643^648. [25] Bray, R.C. (1975) Molybdenum iron-sulfur £avin hydroxylases and related enzymes. In: The Enzymes (Boyer, P.D., Ed.), pp. 300^419. Academic Press, New York.
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The redox centers in the molybdo iron-sulfur £avoprotein CO dehydrogenase from the thermophilic carboxidotrophic bacterium Pseudomonas thermocarboxydovorans Petra Ha«nzelmann, Bettina Hofmann, Sabine Meisen, Ortwin Meyer * Lehrstuhl fu«r Mikrobiologie, Universita«t Bayreuth, Universita«tsstraMe 30, D-95440 Bayreuth, Germany Received 5 March 1999; received in revised form 4 May 1999; accepted 4 May 1999
Abstract The redox centers in the molybdo iron-sulfur flavoprotein CO dehydrogenase from the thermophilic bacterium Pseudomonas thermocarboxydovorans were identified and characterized by electron paramagnetic resonance (EPR). One mol of the 279-kDa dimer contained 1.9 mol of Mo, 2.2 mol of FAD, 6.9 mol of Fe and 6.7 mol of labile sulfide. The molybdenum cofactor is composed of a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and the Mo ion. EPR spectroscopy revealed signals typical for Mo(V), FADH , and type I and type II [2Fe-2S] centers. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Carbon monoxide ; Carboxidotrophic bacterium; CO dehydrogenase; Molybdenum cofactor; Molybdopterin-cytosine dinucleotide
1. Introduction The CO dehydrogenases of the two mesophilic bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudo£ava are molybdo iron-sulfur £avoproteins [1^4]. The enzyme from O. carboxidovorans is a 273.4-kDa dimer of LMS heterotrimers [1,2,5] î and its crystal structure has been resolved at 2.2 A [6]. The L subunit is the molybdoprotein and contains a 1:1 mononuclear complex of a Mo ion and molybdopterin-cytosine dinucleotide (MCD). The M subunit is the £avoprotein and carries the FAD. The
* Corresponding author. Tel.: +49 (921) 552729; Fax: +49 (921) 552727; E-mail: [email protected]
S subunit is the iron-sulfur protein and carries a type I and a type II [2Fe-2S] center. The CO dehydrogenase from the thermophile Pseudomonas thermocarboxydovorans has the same subunit composition [7,8] as the enzyme from the mesophile O. carboxidovorans [1,2,5]. The CO dehydrogenases are members of the sequence family of molybdenum hydroxylases [9]. The enzyme subunits L, M and S of O. carboxidovorans and the subunits A, B and C of P. thermocarboxydovorans show signi¢cant sequence homologies [5,8]. The enzyme from P. thermocarboxydovorans has been identi¢ed as an iron-sulfur £avoprotein containing 2 mol of £avin and nearly 8 mol of Fe and 8 mol of acid labile sulfur per mol of enzyme [7]. The £avin and the iron-sulfur centers have not been analyzed. Although 0.7 mol
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 2 3 0 - X
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of Mo per mol of CO dehydrogenase has been demonstrated, no attempts have been made to study the Mo center by electron paramagnetic resonance (EPR) spectroscopy. The molybdenum cofactor of the CO dehydrogenase from P. thermocarboxydovorans has not been isolated and the type of pterin engaged in the coordinative binding of the Mo ion is not known. That the pterin will be MCD [8] has been assumed from the presence of MCD in the molybdenum cofactor of the CO dehydrogenases from di¡erent bacteria [3], as well as from the similarities between the sequence 712L GGGFGNK718L on the large polypeptide (CutA) of the CO dehydrogenase from P. thermocarboxydovorans and the consensus sequence GXGX2 G for dinucleotide binding [10]. However, the sequences of pterin contacting or conserved segments, e.g. identi¢ed in the structurally characterized Mop protein from Desulfovibrio gigas [11], alone do not permit a solid conclusion on the type of pterin present in a protein. In this paper we report on the identi¢cation and characterization of the redox centers in CO dehydrogenase from P. thermocarboxydovorans.
drophobic interaction chromatography on Source 15 ISO (Pharmacia) and dye ligand a¤nity chromatography on Dyematrex red A (Millipore). The oxidation of CO by CO dehydrogenases was assayed spectrophotometrically [14]. One unit of CO dehydrogenase activity is de¢ned as the amount catalyzing the reduction of 1 Wmol 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride min31 at 30³C.
2. Materials and methods
2.4. Miscellaneous methods
2.1. Bacterial strains and cultivation
Analytical PAGE was carried out on a discontinuous system [17] employing a 5% stacking gel and a 7.5% running gel (native PAGE) or a 7.5% stacking gel and a 12% running gel (SDS-PAGE). Gels were stained for protein with Coomassie brilliant blue G250. The N-termini of subunits were sequenced as described [18]. The method of Bradford [19] was employed for the estimation of soluble proteins. Homogeneous CO dehydrogenases were also quantitated by their absorption at 450 nm employing a millimolar extinction coe¤cient at 450 nm of 72 mM31 cm31 and molecular masses of 283.121 kDa (P. thermocarboxydovorans [8]) or 277.436 kDa (O. carboxidovorans [5]). Analyses of Fe [20], Mo [21] and acid labile sulfur [22] followed published procedures. X-band EPR spectra were recorded on a Bruker EMX spectrometer equipped with a ESR 900 helium cryostat (Oxford Instruments) under the experimental conditions described [4,23].
Bacterial cell mass was produced with CO as the substrate under chemolithoautotrophic conditions in 70-l fermenters ¢lled with 50 l of a mineral medium of pH 7.2 plus trace element solution TS2 containing ammonium Fe(III) citrate (0.1 g l31 ) and ammonium chloride (5 g l31 ) [12]. O. carboxidovorans OM5 (DSM 1227) was grown at 30³C [12] and P. thermocarboxydovorans strain C2 (NCIB 11893) at 50³C [13]. 2.2. Puri¢cation and assay of CO dehydrogenase Puri¢cation of the CO dehydrogenases from P. thermocarboxydovorans [7] or O. carboxidovorans [4] basically followed published protocols and involved precipitation of nucleic acids with 3 mg of protamine sulfate per g of bacterial cell mass, anion exchange chromatography on Source 30 Q (Pharmacia), hy-
2.3. Analysis of pterins, nucleotides and £avins Molybdopterin (MPT) dinucleotides in CO dehydrogenases were analyzed by oxidation with I2 /KI [15]. Carboxamidomethylation of pterins was with iodoacetamide after extraction from CO dehydrogenase with SDS [16] followed by isocratic reversed phase HPLC [4]. FAD, extracted from CO dehydrogenase with SDS, was analyzed by HPLC [4]. FAD was also analyzed spectrophotometrically in supernatants of TCA precipitates [1]. Nucleotides were released from MCD or FAD by hydrolysis of CO dehydrogenase with sulfuric acid followed by HPLC analysis [4].
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3. Results and discussion 3.1. Puri¢cation of CO dehydrogenase and some properties CO dehydrogenase was puri¢ed from P. thermocarboxydovorans about 9-fold with a yield of 22% and a speci¢c activity of 3.1 U mg protein31 . The homogeneity of the preparations is apparent from a single band upon native PAGE (Fig. 1A, lane 2) and the appearance of only the three enzyme polypeptides upon SDS-PAGE (Fig. 1B, lane 4). The mobilities of the CO dehydrogenases from P. thermocarboxydovorans and O. carboxidovorans on native PAGE (Fig. 1A) were in accordance with the molecular masses of (kDa) 279.174 [8] or 273.532 [5], respectively, calculated from the subunit amino acid composition. SDS-PAGE shows that the CO dehydrogenases from both sources have the same (LMS)2 subunit structure (Fig. 1B).
Fig. 2. Comparison of the UV/Vis absorption spectra of air-oxidized CO dehydrogenases (1.33 WM, in 50 mM Hepes, pH 7.2). Sources: P. thermocarboxydovorans, solid line; O. carboxidovorans, dotted line.
The N-termini of the P. thermocarboxydovorans CO dehydrogenase A subunit (NAPLSDREKA), B subunit (MIPPAFAYHA) and C subunit (SKHIVSMTVN) were identical to the sequences reported of CutA, CutB and CutC, respectively [8]. The enzyme has a typical CO dehydrogenase UV/ Vis absorption spectrum exhibiting a protein and cofactor absorption peak at 276 nm, FAD absorption maxima at 386 nm and around 450 nm plus absorptions of the [2Fe-2S] centers around 325, 420, 465 and 550 nm (Fig. 2, solid line). 3.2. Identi¢cation of a mononuclear 1:1 Mo-MCD complex
Fig. 1. Homogeneity and polypeptides of CO dehydrogenase. The CO dehydrogenases puri¢ed from O. carboxidovorans (lanes 1 and 3) or P. thermocarboxydovorans (lanes 2 and 4) were subjected to native (A) and SDS-PAGE (B). Native gels, 50 Wg protein; SDS gels, 70 Wg protein.
CO dehydrogenase from P. thermocarboxydovorans contained 1.94 þ 0.08 mol of Mo mol enzyme31 (three determinations). Iodine oxidation of the enzyme produced a compound with a £uorescence excitation maximum at 380 nm and an emission maximum at 460 nm indicative of form A MPT [24]. The relative £uorescence yields of form A MPT from CO dehydrogenase of P. thermocarboxydovorans (89.1 þ 14.6%) or O. carboxidovorans (set at 100%) indicate that both enzymes contain the same amounts of MPT. HPLC analysis of CO dehydrogenase treated with sulfuric acid revealed 1.91 þ 0.10 mol of 5P-CMP mol enzyme31 (three determinations), which was identi¢ed on the basis of co-elution
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with authentic 5P-CMP at 7.4 min (Fig. 3A). The contents of Mo, 5P-CMP and MPT indicate a mononuclear 1:1 Mo-MCD complex in the CO dehydrogenase from P. thermocarboxydovorans. Direct proof for the presence of MCD was obtained from alkylation of the enzyme with iodoacetamide under reducing conditions. Upon HPLC the eluting compound at 10.1 min was identi¢ed as di(carboxamidomethyl)molybdopterin-cytosine dinucleotide [di(cam)MCD] on the basis of its characteristic UV/Vis absorption spectrum with maxima at 278 nm and 367 nm, an OD278 /OD367 ratio of 3.3 and co-elution with authentic di(cam)MCD (Fig. 3B). 3.3. Characterization of the metal centers by EPR EPR spectroscopy of dithionite-reduced CO dehydrogenase from P. thermocarboxydovorans revealed at 120 K a rhombic Mo(V) signal with g factors of g1 = 1.978, g2 = 1.967 and g3 = 1.954 (gav = 1.966) and a line width of 7 mT (Fig. 4A, a). In addition to the intense signal of the 96 Mo isotope (74.5% natural abundance) without a nuclear spin, weaker signals arising from hyper¢ne interaction with the 95 Mo and 97 Mo nuclei, both with a nuclear spin of I = 5/ 2 and with natural abundances of 15.9% and 9.6%, are visible (Fig. 4A, a). Very similar Mo(V) signals have been reported for the CO dehydrogenases from O. carboxidovorans [23] and H. pseudo£ava [4]. Airoxidized CO dehydrogenase was EPR silent at 120 K (Fig. 4A, b), referring to a Mo(VI). Upon reduction with CO, CO dehydrogenase revealed at 120 K an isotropic signal (g = 2.005), typical of an organic radical, and additional periodically repeating signals (Fig. 4A, c). At 1 mW microwave power the signal at g = 2.005 has a peak to peak line width of 0.21 mT, indicative of the neutral FADH semiquinone radical [25]. The FADH radical has a maximum signal intensity at about 120 K and is saturated at P1=2 = 0.5 mW (Fig. 5). The additional periodically repeating EPR lines were saturated at P1=2 = 20 mW (Fig. 5). These signals showed maximum intensities below 75 K; the exact temperature could not be determined because below 70 K the [2Fe-2S] EPR signals became apparent. The crystal structure of CO dehydrogenase from O. carboxidovorans shows a catalytic essential selenium atom in a distance of î to the Mo ion [6]. The selenium is bound to 3.7 A
Fig. 3. A: HPLC elution of the 5P-nucleotides obtained through acid hydrolysis of the CO dehydrogenases from P. thermocarboxydovorans (1.71 nmol, trace I) or O. carboxidovorans (0.85 nmol, trace II) and the corresponding spectra of the materials eluting at 7.4 min and 17.4 min of trace I (inset). B: HPLC elution of the alkylated pterins isolated from the CO dehydrogenases from P. thermocarboxydovorans (14 nmol, trace I) or O. carboxidovorans (7 nmol, trace II) and the spectra of the materials eluting at 10.1 min (inset). C: HPLC elution of the £avins extracted with SDS from the CO dehydrogenases from P. thermocarboxydovorans (3.74 nmol, trace I) or O. carboxidovorans (1.87 nmol, trace II) and the spectra of the materials eluting at 22.8 min (inset).
the cysteine388L residue which is situated in a loop (384L VAYRCSFR391L ) in the active site. The P. thermocarboxydovorans CO dehydrogenase has the same sequence (378A VAYRCSFR385A ), including cysteine382A on CutA. Therefore, we ascribe the repeating EPR lines in the spectrum of Fig. 4A, c to a Mo/ Se center, produced upon substrate binding/oxidation.
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Fig. 4. Mo(V) and iron-sulfur EPR spectra of CO dehydrogenase from P. thermocarboxydovorans (35 WM, in 50 mM Hepes, pH 7.2). Reduction under anoxic conditions was with 9.5 mM dithionite or CO. Instrument settings : microwave frequency, 9.47 GHz; modulation amplitude, 1 mT. A: Spectra originating from Mo(V) were recorded at 120 K with a microwave power of 10 mW. a, dithionite-reduced ; the parts of the spectra at high and low g factors are shown with the vertical scale increased 5-fold ; b, air-oxidized ; c, CO-reduced. B: Iron-sulfur EPR was at 16 K with a microwave power of 200 mW or at 49 K with a microwave power of 10 mW. d, 16 K, dithionitereduced ; e, 16 K, air-oxidized ; f, 16 K, CO-reduced ; g, 49 K, dithionite-reduced ; h, 49 K, air-oxidized ; i, 49 K, CO-reduced. The numbers in the spectra indicate the g values.
P. thermocarboxydovorans CO dehydrogenase contained 6.90 þ 0.18 mol of Fe and 6.72 þ 0.18 mol of acid labile sulfur mol enzyme31 (three determinations). Dithionite-reduced CO dehydrogenase from P. thermocarboxydovorans showed at temperatures below 60 K rhombic EPR signals of two di¡erent [2Fe-2S] centers (Fig. 4B, d, g). As de¢ned with xanthine oxidase, and according to the convention in the literature [25], the rhombic [2Fe-2S] signal with the smaller g anisotropy is termed type I [g1 = 2.024, g2 = 1.945, g3 = 1.910, gav = 1.960 (Fig. 4B, g)], and that with the larger g anisotropy is termed type II [g1 = 2.158, g2 = 1.978, g3 = 1.895, gav = 2.010 (Fig. 4B, d)]. At 49 K almost only the signals of the type I center were detectable (Fig. 4B, g). At 16 K a combined EPR spectrum of the signals originating from the type I and the type II centers was visible (Fig. 4B, d). In addition, features due to the presence of Mo(V) at 49 K (g = 1.977 and g = 1.970) were present (Fig. 4B, g). The air-oxidized enzyme was EPR silent (Fig. 4B, e, h). The CO-reduced enzyme revealed at 16 K and 49 K signals of the type I and type II [2Fe-2S] centers (Fig. 4B, f, i).
Fig. 5. Microwave power saturation behavior of CO-reduced FADH and Mo(V) signals at 120 K. Spectra were recorded using the conditions given in Fig. 4A, except that the microwave power (P) was varied over the range of 0.01^200 mW. The signal intensities (S) of the following spectral features were measured : for FADH , the overall signal amplitude above and below baseline; for Mo(V), the overall signal amplitude of g1:946 above and below baseline. The determination of P1=2 was as described [23].
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3.4. Presence of FAD Trichloroacetic acid supernatants of CO dehydrogenase from P. thermocarboxydovorans revealed 2.20 þ 0.12 mol of £avin mol enzyme31 (three determinations), which was identi¢ed as FAD on the basis of the appearance of 1.76 þ 0.25 mol of 5P-AMP mol enzyme31 (three determinations) at 17.4 min upon HPLC of acid hydrolysates of CO dehydrogenase (Fig. 3A). HPLC analysis of CO dehydrogenase unfolded with SDS revealed a single compound absorbing at 450 nm which was identi¢ed as FAD on the basis of its characteristic UV/Vis absorption spectrum and co-elution with authentic FAD (Fig. 4C). The presence of FAD in CutB of CO dehydrogenase from P. thermocarboxydovorans is apparent from the sequences 32B AGGHS36B and 111B TIGG114B which have been identi¢ed in the high resolution structure of the CO dehydrogenase from O. carboxidovorans as £avin-binding [6].
[5]
[6]
[7]
[8]
[9] [10]
[11]
Acknowledgements We thank Dr. Heinz Faulhammer (Universita«t Bayreuth) for the determination of N-terminal amino acid sequences. This work was ¢nancially supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt am Main, Germany).
[12]
[13]
[14]
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P. Ha«nzelmann et al. / FEMS Microbiology Letters 176 (1999) 139^145 quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248^ 254. [20] Fish, W.W. (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol. 158, 357^364. [21] Cardenas, J. and Mortenson, L.E. (1974) Determination of molybdenum and tungsten in biological materials. Anal. Biochem. 60, 372^381. [22] Fogo, J.K. and Popowsky, M. (1949) Spectrophotometric determination of hydrogen sul¢de. Anal. Chem. 21, 732^734.
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[23] Bray, R.C., George, G.N., Lange, R. and Meyer, O. (1983) Studies by e.p.r. spectroscopy of carbon monoxide oxidases from Pseudomonas carboxydovorans and Pseudomonas carboxydohydrogena. Biochem. J. 211, 687^694. [24] Meyer, O. and Rajagopalan, K.V. (1984) Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. J. Bacteriol. 157, 643^648. [25] Bray, R.C. (1975) Molybdenum iron-sulfur £avin hydroxylases and related enzymes. In: The Enzymes (Boyer, P.D., Ed.), pp. 300^419. Academic Press, New York.
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