Bacteria ORTWIN MEYER'* and K

JOURNAL OF BACTERIOLOGY, Feb. 1984, p. 643-648 Vol. 157, No. 2 0021-9193/84/020643-06$02.00/0 Copyright C 1984, American Society for Microbiology Molybdopterin in Carbon Monoxide Oxidase from Carboxydotrophic Bacteria ORTWIN MEYER'* AND K. V. RAJAGOPALAN2 Institut fiar Mikrobiologie der Universitat Gottingen, D-3400 Gottingen, Federal Republic of Germany, and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 277102 Received 2 September 1983/Accepted 4 November 1983 The carbon monoxide oxidases (COXs) purified from the carboxydotrophic bacteria Pseudomonas carboxydohydrogena and Pseudomonas carboxydoflava were found to be molybdenum hydroxylases, identical in cofactor composition and spectral properties to the recently characterized enzyme from Pseudomonas carboxydovorans (0. Meyer, J. Biol. Chem. 257:1333-1341, 1982). All three enzymes exhibited a cofactor composition of two flavin adenine dinucleotides, two molybdenums, eight irons and eight labile sulfides per dimeric molecule, typical for molybdenum-containing iron-sulfur flavoproteins. The millimolar extinction coefficient of the COXs at 450 nm was 72 (per two flavin adenine dinucleotides), a value similar to that of milk xanthine oxidase and chicken liver xanthine dehydrogenase at 450 nm. That molybdopterin, the novel prosthetic group of the molybdenum cofactor of a variety of molybdoenzymes (J. Johnson and K. V. Rajagopalan, Proc. Natl. Acad. Sci. U.S.A. 79:6856-6860, 1982) is also a constituent of COXs from carboxydotrophic bacteria is indicated by the formation of identical fluorescent cofactor derivatives, by complementation of the nitrate reductase activity in extracts ofNeurospora crassa nit-i, and by the presence of organic phosphate additional to flavin adenine dinucleotides. Molybdopterin is tightly but noncovalently bound to the protein. COX, sulfite oxidase, xanthine oxidase, and xanthine dehydrogenase each contains 2 mol of molybdopterin per mol of enzyme. The presence of a trichloroacetic acid-releasable, so-far-unidentified, phosphorous-containing moiety in COX is suggested by the results of phosphate analysis.

Carbon monoxide serves as the sole carbon and energy carboxydotrophic bacteria (3). Drake and co-workers (10, source under aerobic conditions for carboxydotrophic bacte- 11) showed that the enzyme from C. thermoaceticum is also ria (6, 14, 29, 32, 35, 44, 52). The metabolism of carboxydo- a metallo iron-sulfur protein, containing 2 nickels, 1 zinc, 11 trophs is facultatively chemolithoautotrophic, and they are irons, and 14 acid-labile sulfurs per molecule. Molybdenum exceptional within the physiological grouping of chemolitho- or flavins are not contained in that enzyme, and the iron- trophs in being able to generate their own CO2 for assimila- sulfur centers are of the Fe4S4 type (39). tion. Most of the work on carboxydotrophs has been done In this paper, we provide further and direct evidence for with Pseudomonas carboxydovorans (32, 35). our hypothesis (6, 30, 35, 43) that COXs from P. carboxydo- CO is cooxidized by the methanotrophic bacteria, e,g., vorans, Pseudomonas carboxydohydrogena, Pseudomonas Pseudomonas methanica, Methylosinus trichosporium, and carboxydoflava, and probably from all other carboxydotro- Methylococcus capsulatus, owing to the broad specificity of phic bacteria are extremely similar enzymes. They are novel the methane monooxygenase complex (4). molybdoproteins, and we report here on the presence of the Under anaerobic conditions, methanogenic bacteria, e.g., molybdenum cofactor common to nitrate reductase, xan- Methanobacterium thermoautotrophicum, can reduce CO thine oxidase (XO), xanthine dehydrogenase (XDH), and with H2 to form methane (7). Acetogenic bacteria, e.g., sulfite oxidase (SOX) in COXs from carboxydotrophic bac- Clostridium thermoautotrophicum, Clostridium pasteur- teria also. Recent studies on the structure of an oxidized, ianum, or Clostridium thermoaceticum, form acetate and fluorescent derivative (form A) of the native cofactor ob- CO2 from CO (12, 23, 45). Sulfate-reducing bacteria, e.g., tained by denaturation of molybdoenzymes in the presence Desulfovibrio desulfuricans (49-51), and phototrophic bacte- of iodine have established that molybdenum cofactor con- ria in the dark, e.g., Rhodopseudomonas gelatinosa or tains a novel pterin (17, 18, 42), and a structure for the native Rhodospirillum rubrum (46, 47), form CO2 and H2 from CO molybdopterin (MPT), defined as the organic, metal-free under anaerobic conditions. component of the molybdenum cofactor, has been proposed Enzymes specifically catalyzing the oxidation of CO have by Johnson and Rajagopalan (20). The present investigation been most fully characterized in P. carboxydovorans (3, 30, was undertaken to determine whether COX from carboxydo- 33, 34, 43) and C. thermoaceticum (8, 10, 11, 38, 39). Both trophs contains this pterin structure. Evidence is presented enzymes catalyze the same oxidation reaction: CO + H20 that 2 mol of the novel MPT are contained per mol of COX, CO2 + 2 e- + 2 H+. However, they transfer the electrons to SOX, XDH, and XO. different acceptors and are remarkably dissimilar in other respects also. The enzyme from P. carboxydovorans is a MATERIALS AND METHODS molybdenum-containing iron-sulfur flavoprotein as men- Materials. Sodium dodecyl sulfate (SDS) was obtained tioned above (30). Electron paramagnetic resonance mea- from Schwarz/Mann; guanidine hydrochloride was pur- surements revealed the presence of two different iron-sulfur chased from Heico (Whittaker Corp.); Sephadex G-15 was centers of the Fe2S2 type in CO oxidase (COX) from from Pharmacia Fine Chemicals, and quaternary aminoethyl Sephadex (Q-25) was obtained from Sigma Chemical Co, * Corresponding author. The quaternary aminoethyl Sephadex u~s equilibrated with 643 644 MEYER AND RAJAGOPALAN J. BACTERIOL.

1 M ammonium acetate and then washed extensively with TABLE 1. Spectral properties of COXs from diverse sources water. Millimolar extinc- Growth of bacterial strains. The following bacterial strains tion coefficient at were used: P. carboxydovorans OM5 (DSM1227). (32, 35), P. Absorption ratios 450 nm on the (Seliberia) carboxydohydrogena Z-1062 (DSM1083) (31, 35, Bacterial strain basis of 52)- and P. carboxydoflava Z-1107 (DSM1084) (26, 35, 52). FAD Dry Bacteria were grown CO-autotrophically in mineral medium A280A450 A45JA550 content wt under described conditions (32, 35). Ammonium chloride P. carboxydovorans 5.0 3.0 71.9 70.8 served as the nitrogen source, and the supplement of sodium P. carboxydohydrogena 5.0 3.1 71.4 69.7 molybdate in the trace element solution was increased to 900 P. carboxydoflava 5.3 3.0 71.6 67.2 mg/liter (35). Cells grown to the late exponential phase were harvested by centrifugation, and packed cells were frozen and stored at -20°C. Purification of molybdoenzymes. COX was prepared from cifically bound phosphates, all molybdoenzymes were carboxydotrophic bacteria as described previously (3, 30, passed over a Sephadex G-25M column (PD-10), and dia- 34). XDH and SOX were purified from chicken livers as lyzed twice against 5 liters of 10 mM Tris buffer (pH 7.4) for described (24), and XO was prepared from milk by the 12 h. Enzyme samples (0.3 ml or less) in the presence or method of Waud et al. (48). All enzymes were highly pure absence of 5 to 10 nmol of P1 as an internal standard were and of complete cofactor composition as indicated by single carefully layered on the bottom of small Pyrex test tubes and bands in nondenaturing polyacrylamide gel electrophoresis, brought to dryness in an aluminum block by increasing the maximum specific activities and by absorption ratios (Table temperature slowly to 120°C. After cooling the tubes to room 1) typical of highly pure molybdoenzymes of complete temperature, 20 RI of 50% (vol/vol) concentrated sulfuric cofactor composition (5, 40). acid in water and 20 ,ul of 70% (vol/vol) of perchloric acid Determination of apparent molecular weight, dry weight, (PCA) were added. The tubes were heated to 140°C in the and FAD content. Molecular weight determinations were aluminum block and maintained at that temperature for 30 performed by high-pressure liquid chromatography on a min. Then, 10 RI of 30% (vol/vol) H202 was added, and the sizing column (TSK SW-3000 from Kratos Analytical) with samples were oxidized to completion by shaking the tubes in 100 mM phosphate buffer (pH 7.0) as the liquid phase. XDH the open flame of a bunsen burner till all of the brownish (chicken liver), catalase, alcohol dehydrogenase, serum color had disappeared. After the addition of 260 RI1 of water, albumin (bovine), ferritin, and cytochrome c served as phosphate was quantitated by the method of Ames (1). molecular weight standards. The high-pressure liquid chro- Under these conditions, 10 nmol of phosphate gave an matography system consisted of a Constametric III pump absorbance reading at 820 nm of 0.260, and the recovery of (LDC/Milton Roy) and a Kratos 773 UV/VIS detector. Pi added to protein samples was at least 95%. The nominal dry weight of the enzymes was obtained by Alkaline phosphatase from chicken intestines was from drying a measured volume of known protein and flavin Worthington Diagnostics, and cleavage was carried out on adenine dinucleotide (FAD) content in an oven at 105°C to a samples at pH 8 to 9 in the presence of 7.5 mM MgCI2. constant weight. Before this procedure, the enzymes were Determination of molybdenum. Molybdenum was quanti- passed over a PD-10 column (Pharmacia) and dialyzed twice tated by atomic absorption spectroscopy by using a Perkin against 4 liters of distilled water for 15 h. Corrections were Elmer 107 spectrometer equipped with a graphite rod assem- made for the dry weight by using the water ofthe last dialysis bly. step. Samples were brought to constant weight by incubation in the oven for 12 h, followed by cooling in a vacuum RESULTS desiccator containing drying gel. Spectral properties of COXs from different sources. COX FAD was extracted with trichloroacetic acid (TCA) and was isolated from the carboxydotrophic bacteria P. carboxy- quantitated from its absorbance at 450 nm in the neutralized dovorans, P. carboxydohydrogena, and P. carboxydoflava. solution (30). Absorption spectra of the three enzymes were found to be Spectra. UV and visible absorption spectra were recorded identical and were very similar to those of milk XO and on a Shimadzu UV 2000 spectrophotometer- (Fig. 1). En- chicken liver XDH. The spectra were characterized by the zyme concentrations were determined by use of millimolar typical broad shoulder at 550 nm and a peak at 450 nm (Fig. extinction coefficients of 72 at 450 nm for COX, XDH, and 1). Like the other highly purified metalloflavoproteins the XO (5, 35) or of 216 at 413.5 nm for SOX (40). Fluorescence three COXs exhibited ratio of 5 for absorbance at 280 nm to spectra were obtained with an Aminco-Bowman SPF spec- absorbance at 450 nm (A28d/A450) and a A450/A550 ratio of 3 trofluorometer. All fluorescence spectra were uncorrected. (Table 1). Oxidation of native molybdenum cofactor. Enzyme-bound The tnillimolar extinction coefficients at 450 nm on the MPT was converted to fluorescent oxidation products by basis of FAD content and of dry weight of the three COXs being boiled at pH 2.5 for 25 min in the absence (form B) or were similar and amounted to 67 to 72 (Table 1). Nearly presence of KI plus I2 (form A) by the method of Johnson identical values have been reported for other metalloflavo- and Rajagopalan (20). Fluorescence was measured in cu- proteins (5, 41). vettes of 1-cm light path after the addition of NH40H to a 1 The preparations of COXs used throughout this study N final concentration by using an Aminco-Bowman spectro- contained 1.9 to 2.1 mol of FAD per mol of enzyme, and the fluorometer. molybdenum content was between 1.8 and 2.0 mol of Mo per Molybdenum cofactor activity was assayed by measuring 2 mol of FAD. The specific CO -_ methylene blue and reconstitution of nitrate reductase activity in extracts of NADH -* methylene blue activities of the three enzymes induced cells of the Neurospora crassa nit-i mutant (2, 36, were also in the same order of magnitude. The COXs 37). comigrated with chicken liver XDH upon chromatography Phosphate determination. To decrease the level of unspe- on a high-pressure liquid chromatography sizing column, VOL. 157, 1984 MPT IN COX FROM CARBOXYDOTROPHS 645

second (acidic species) eluting with 0.01 N HCI. The fluorescent fractions (peak 2) were pooled, evaporated in a vacuum, and redissolved in 1 N NH40H. The resulting fluorescence spectra were characteristic ofform A, and the two enzymes exhibited practically the same specific fluorescence (Fig. 3). Quantitation of MPT. It had been concluded from the presence of 2.2 mol of organic phosphate per mol of SOX and the presence of 0.81 mol of organic phosphate per mol of 002 isolated form A that native MPT contains a single phosphate go0.2 tXO -XDH and that 2 mol of MPT are associated per mol of SOX (42). The equivalence of organic phosphate and MPT offered the possibility to quantitate the pterin content of molybdoenzymes by phosphate determination. The content of organic 0.1 phosphate in native molybdoenzymes as well as in supernatants and pellets after precipitation with TCA or PCA was determined in several preparations of COX, SOX, XO, and XDH (Table 2). The presence of 2 mol of organically bound phosphate per mol of SOX has been confirmed; the presence 300 400 500 600 of 6 mol of organic phosphate per mol of XDH or XO is Wavelength Inml explained by a molar content of 2 MPTs and 2 FADs. FIG. 1. Visible absorption spectra of oxidized molybdenum hy- Compared with XDH and XO, an additional 2 mol of organic droxylases. All enzymes were in 10 mM Tris-hydrochloride buffer phosphate were present in the three CO oxidases; this made (pH 7.2). The concentrations of COXs from P. carboxydovorans, P. it impossible to quantitate the pterin in COX on the basis of carboxydohydrogena, and P. carboxydoflava were matched to give the same absorbancy at 450 nm. XDH, chicken liver XDH; XO, milk FAD and phosphate content alone. XO. (Reprinted from Meyer and Schlegel, Annu. Rev. Microbiol. Precipitation of COX with TCA or PCA was found to 37:277-310, 1983, by permission of Annual Reviews, Inc.) release the flavin but not the pterin. Therefore, phosphate determinations were also done on TCA or PCA supernatants and on the corresponding pellets of molybdoproteins (Table 2). The four enzymes revealed (per mole) 1.44 to 2.2 mol of indicating a molecular weight of 300,000 under these condi- organic phosphate in the pellet, suggesting the presence of 2 tions. mol of pterin in COX also. The 4 mol of organic phosphate Identification of the molybdenum cofactor. The existence found in the supernatants of XDH and XO can be ascribed to of a molybdenum cofactor common to nitrate reductase, the presence of 2 mol of FAD. The additional 2 mol of XO, XDH, and SOX has been firmly established by studies organic phosphate present in COX appeared in the TCA or from several laboratories. Acidification or treatment of COX PCA supernatant, suggesting the existence of an as yet with 6 M guanidine or 1% (wt/vol) SDS liberated a cofactor unidentified, noncovalently bound, organic phosphorous capable of complementing apo-nitrate reductase in extracts compound. The phosphate of this unknown material was not of the Neurospora crassa nit-I mutant (35). On a molar cleaved by alkaline phosphatase, and the radioimmunoassay basis, identical complementing activities were found for for cyclic AMP in neutralized TCA supernatants of COX COX, XDH, XO, and SOX (R. V. Hageman and K. V. was negative. Controls, established by the addition of cyclic Rajagopalan, personal communication), suggesting the pres- AMP to the samples, were positive. ence of the same amount of Mo cofactor in these enzymes. Finally, it should be mentioned that the Pi content of TCA The molybdenum cofactor isolated from chicken liver or PCA supernatants never exceeded 0.2 mol per mol of SOX and XDH has been identified as a novel pterin. The enzyme. However, even extensively dialyzed native XDH probable chemical nature of the pterin and its major func- always liberated exactly 2 mol of Pi under these conditions. tional groups have been identified through studies of inac- tive, oxidized, degradation products (17, 18, 20, 42). Oxida- tion of the three COXs also revealed compounds exhibiting 2.1 the blue pterin-like fluorescence. When COX was oxidized with iodine and the resulting fluorescence was measured in 1 N ammonia, the excitation and emission spectra were identical to those of SOX, XDH, and XO treated the same way (Fig. 2). This oxidation product of the native cofactor has 03 been termed form A (18, 20). The relative amounts of 0 fluorescence formed per mole of enzyme were more or less 0L the same for the three COXs and the other molybdoenzymes tested, even under these crude conditions (Table 2). When the oxidation in boiling acid (pH 2.5) was done in 200 300 400 500 the absence of iodine, the three COXs produced identical Wavelength I nm I amounts of fluorescence, characteristic of form B. FIG. 2. Fluorescence spectra ofform A derivatives of the native molybdenum cofactor. Enzyme samples were denatured in boiling Same amounts of COX from P. carboxydovorans and XO acid (pH 2.5) in the presence of KI and 12, and the fluorescence from bovine milk were denatured in the presence of iodine, spectra were recorded in 1 N ammonia. The spectra refer to (from and the fluorescent material was purified on the anion top to bottom): SOX (chicken liver), XDH (chicken liver), COX (P. exchanger quaternary aminoethyl Sephadex; under these carboxydovorans), and XO (cow milk). The excitation spectra conditions, two predominant fluorescent species appeared, (emission set at 460 nm) are on the left, and the emission spectra one eluting in 0.01 N acetic acid (neutral species), and the (excitation at 380 nm) are on the right. 646 MEYER AND RAJAGOPALAN J. BACTERIOL.

TABLE 2. Quantitation of molybdenum cofactor in different molybdoenzymes by phosphate analysis and by oxidation with iodine to form A Mean organic phosphate contenta Fluorescence Enzyme Pellet Supernatant Sum Total at 455 nmb Cox (P. carboxydovorans) 1.98 5.74 7.72 7.98 29.2 (P. carboxydohydrogena) 7.96 27.4 (P. carboxydoflava) 8.1 31.8 Sox (chicken liver) 1.44 0.5 1.94 2.0 35.2 XDHc (chicken liver) 2.22 3.67 5.89 6.24 26.7 xo (milk) 1.74 4.22 5.96 6.04 29.7 a Values are per mole of enzyme. Samples were ashed as described in the text. All values are corrected for inorganic phosphate. Values for the pellet and supernatant were after precipitation of enzyme samples (maximum 0.3 ml) with 5 to 10% (vol/vol) TCA or PCA. Sums indicate organic phosphate contained in pellets and supernatants. Totals indicate the content of organic phosphate of the native enzymes after ashing. b Relative fluorescence per micromole of enzyme was measured in 1 N ammonia at an excitation wavelength of 380 nm. c XDH consistently revealed 2 mol of Pi.

These observations can be interpreted by the presence of num hydroxylases identical to chicken liver XDH and milk two specific binding sites for Pi in XDH that are not evident XO in their gross molecular properties. The COXs from the in the other molybdoenzymes. three bacteria contain 2 FADs and 2 Mos per molecule, and Hydrolysis of cofactor phosphate. Based on their rates of the ratio of A45G/A550 = 3 is indicative for the presence of 4 hydrolysis, organic phosphates have been separated into two FeS entities per FAD (41). The enzymes from P. carboxydo- main groups. Those which are hydrolyzed in 1 N hydrochlo- vorans and P. carboxydohydrogena have been shown to ric acid at 100°C in 7 min (27) or in 1 N sulfuric acid in 15 min contain two different 2Fe-2S centers exhibiting the same (28) are usually called labile. Those which are not hydro- electron paramagnetic resonance properties as the xanthine lyzed under these conditions are called stable or residual. oxidizing enzymes, and all three enzymes had a Mr of The conditions were selected originally for estimating the 300,000. As with the other purified molybdenum hydroxy- two terminal phosphate groups in ATP. They were applied lases (5), A28d/A450 ratios close to 5 and A45s/Asso ratios close here to determine the type of phosphate linkage in MPT of to 3 reflected complete cofactor composition. Our hypothe- different molybdoenzymes. sis that the COXs from all carboxydotrophic bacteria are Native SOX, as well as pellets of PCA-precipitated COX very similar is based on their identical molecular weight, or XO, were boiled in 1.4 N HCl for 6 h, and Pi was electron-acceptor spectrum, and immunological identity (6, determined in time intervals of 30 min. The different samples 26, 35, 43) and is confirmed for the three different strains exhibited identical time courses of Pi formation, and in all studied here by identical spectral properties and the pres- cases, a plateau was reached at 2 mol of Pi released per mol ence of Mo, FAD, FeS, and MPT. of enzyme after 6 h of incubation. The existence of a molybdenum cofactor common to When COX, SOX, and XO were boiled for 7 min in 1 N nitrate reductase and XDH was first postulated by Pateman HCl or for 15 min in 1 N H2SO4 and analyzed for Pi, none of et al. (37), and convincing evidence for its universality was them revealed more than 0.1 mol of Pi per mol, indicating the provided by the finding of Nason et al. (36) that acid- absence of labile phosphate groups (for a list of compounds, denatured molybdoenzymes from different sources serve as see reference 27) and characterizing all organic phosphate donors of cofactor for the complementation of nitrate reduc- present in these enzymes as stable. Under these conditions tase in Neurospora crassa nit-I extracts. The active cofactor XDH released only its 2 mol of Pi. Pi was also not formed is presumably composed of molybdenum and a reduced form under the conditions of form A production when the molybdoenzymes were boiled in acid (pH 2.5) in the presence of KI and 12. MPT is noncovalently bound to COX. Precipitation of COX, XDH, or XO with TCA or PCA was very effective in releasing the noncovalently bound FAD, but this procedure did not release MPT. However, exposure of COX to 6 M guanidine or 1% (wt/vol) SDS led to the complete release of MPT (Fig. 4), indicating the noncovalent nature of its bond to the protein. In contrast to the molybdoflavoenzymes, appreciable amounts of the pterin of SOX were already released by boiling or TCA precipitation; this indicates that the pterin is more loosely attached to SOX than to COX, Wavelength inmi XDH, or XO. FIG. 3. Form A derivative isolated from COX and XO. Same amounts of COX from P. carboxydovorans (upper curve) or milk DISCUSSION XO (lower curve) were subjected to iodine oxidation, and form A was isolated by means of chromatography on QAE-Sepharose as The work presented here confirms and extends our view described in the text. Fluorescence spectra of purified form A (6, 30, 35, 43) that the COXs from P. carboxydovorans, P. derivatives of the native cofactor were measured in 1 N ammonia, as carboxydohydrogena and P. carboxydoflava are molybde- for the experiment shown in Fig. 2. VOL. 157, 1984 MPT IN COX FROM CARBOXYDOTROPHS 647

in Neurospora crassa is constitutive, and its synthesis is independent of nitrate induction and is not repressed by ammonia. COX is inducible in P. carboxydovorans and P. carboxydohydrogena (6, 33, 35) and is constitutive in P. carboxydoflava (25, 35). Presently, it is not known whether 00 or not the molybdenum cofactor is constitutive in carboxy- os LX oco dotrophs and what the effect of the availability of molybde- ~~~~~~~~w num on cofactor synthesis is. We were recently able to isolate mutants of P. carboxydovorans unable to grow with 0 0~/ CO; an appreciable number of them had lost the capability to form nitrite from nitrate and had acquired resistance to 100 1 /~ mM chlorate. This suggests that these mutants are devoid of the molybdenum cofactor; it is, therefore, likely that they are of the Neurospora crassa nit-i, Aspergillus niger cnx or Escherichia coli chlA type.

0 ACKNOWLEDGMENTS 0 10 20 30 Fraction number O.M. is most grateful to all members of K. V. Rajagopalan's group for their generosity and hospitality during his one-year stay as FIG. 4. Treatment of COX with 6 M guanidine hydrochloride. A a visiting scientist at the Department of Biochemistry, Duke Univer- sample of COX in 100 mM Tris buffer pH 7.8 was incubated in the sity Medical Center. presence of 6 M guanidine hydrochloride at room temperature for 20 The expert technical assistance of Verena GroBe and Ralph Wiley min. The sample was applied to a Sephadex G-15 column and eluted in purifying the molybdoenzymes used in these studies is gratefully with 6 M guanidine in 100 mM Tris pH 7.8 (1 fraction, 0.5 ml). acknowledged. Cofactor was monitored in each fraction by production of 380 This work was supported by Public Health Service grant GM fluorescence. For this purpose 65 ,dl of 1% KI plus 1% 12 in 0.2 M 00091 from the National Institutes of Health to K.V.R. and by grants phosphoric acid were added to each fraction and boiled for 20 min; Me702/1-1 and Me702/1-2 from the Deutsche Forschungsgemeins- fluorescence emission at 460 nm (excitation at 380 nm) was mea- chaft to O.M. sured after mixing of 0.2 ml of this solution with 1 ml of 1 N ammonia. Protein was monitored by measurement of UV absorption LITERATURE CITED after addition of 0.5 ml of guanidine hydrochloride in Tris to each fraction. 1. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8:115-118. 2. Amy, N. K., and K. V. Rajagopalan. 1979. Characterization of molybdenum cofactor from Escherichia coli. J. Bacteriol. of a pterin (15, 17, 18, 20, 42). A possible structure for native 140:114-124. MPT has recently been proposed by Johnson and Rajagopa- 3. Bray, R. C., G. N. George, R. Lange, and 0. Meyer. 1983. Studies by e.p.r spectroscopy of carbon monoxide oxidases lan (20). That the molybdenum cofactor common to molyb- from Pseudomonas carboxydovorans and Pseudomonas car- doenzymes is also a constituent of the COXs from aerobic boxydohydrogena. Biochem. J. 211:687-694. carbon monoxide-oxidizing bacteria is indicated by the abili- 4. Colby, J., H. Dalton, and R. Whittenbury. 1979. Biological and ty of acid-, guanidine- or SDS-treated samples of that biochemical aspects of microbial growth on C, compounds. enzyme to complement nit-I nitrate reductase. The identity Annu. Rev. Microbiol. 33:481-517. of the MPT of SOX, XDH, XO, and COX is supported by 5. Coughlan, M. P., 1980. Aldehyde oxidase, xanthine oxidase and the following arguments. (i) The four different enzymes xanthine dehydrogenase: hydroxylases containing molybde- revealed identical nit-i complementation activities (R. V. num, iron-sulphur and flavin, p. 119-185. In M. P. Coughlan Hageman and K. V. Rajagopalan, personal (ed.), Molybdenum and molybdenum-containing enzymes. Per- communication). gamon Press, Ltd., Oxford. (ii) COX, SOX, XDH, and XO same formed amounts of 6. Cypionka, H., 0. Meyer, and H. G. Schlegel. 1980. Physiological identical, fluorescent degradation products of the native characteristics of various species of strains of carboxydobac- pterin. (iii) The presence of 2 mol of pterin phosphate per teria. Arch. Microbiol. 127:301-307. mol of SOX, XDH, XO, and COX was established on the 7. Daniels, L., G. Fuchs, R. K. Thauer, and G. Zeikus. 1977. basis of total organic phosphate content and by separation of Carbon monoxide oxidation by methanogenic bacteria. J. Bac- pterin from FAD, followed by the determination of organic teriol. 132:118-126. phosphate in the pellet. Conditions effective in releasing FAD 8. Diekert, G., and M. Ritter. 1983. Purification of the nickel from COX, e.g., denaturation by boiling or deprotonization, protein carbon monoxide dehydrogenase of Clostridium ther- 151:41-44. did not liberate the pterin; the latter was released, however, moaceticum. FEBS Lett. 9. Diekert, G. B., E. G. Graf, and R. K. Thauer. 1979. Nickel by treatment with guanidine or SDS, indicating that the requirement for carbon monoxide dehydrogenase formation in pterin is tightly but noncovalently attached to COX. The Clostridium pasteurianum. Arch. Microbiol. 122:117-120. presence of MPT in COXs also provides additional evidence 10. Drake, H. L. 1982. Occurrence of nickel in carbon monoxide for the universality of the molybdenum cofactor in molybdo- dehydrogenase from Clostridium pasteurianum and Clostridium proteins. In contrast, CO dehydrogenase from the anaerobic thermoaceticum. J. Bacteriol. 149:561-566. acetogenic C. thermoaceticum contains nickel (9, 10) and a 11. Drake, H. L., S.-I. Hu, and H. G. Wood. 1980. Purification of noncovalently bound nickel cofactor of Mr 1,357 (38, 39). carbon monoxide dehydrogenase, a nickel enzyme from Clos- Methanopterin (Bo) and carboxydihydromethanopterin tridium thermoaceticum. J. Biol. Chem. 255:7174-7180. 12. Fuchs, G., U. Schnitker, and R. K. Thauer. 1974. (YFC) from Methanobacterium thermoautotrophicum are Carbon monoxide oxidation by growing cultures of Clostridium pasteur- novel pterins involved in methanogenis from CO2 and meth- ianum. Eur. J. Biochem. 49:111-115. anol (21, 22). It can be suspected that they also play a role in 13. Gyure, W. L. 1972. A protein precipitant useful in pteridine and methane formation from CO. purine analysis. Anal. Biochem. 50:309-312. As discussed by Johnson (16), the molybdenum cofactor 14. Hegeman, G. D. 1980. Oxidation of carbon monoxide by bacte- 648 MEYER AND RAJAGOPALAN J. BACTERIOL.

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