Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas Carboxydovorans

JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 811-817 Vol. 137, No. 2 0021-9193/79/02-081 1/07$02.00/0

Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas carboxydovorans

ORTWIN MEYER AND HANS G. SCHLEGEL* Institut fur Mikrobiologie der Universitat, 3400 Gottingen, Federal Republic of Germany Received for publication 26 October 1978

Extracts of aerobically, CO-autotrophically grown cells of Pseudomonas carboxydovorans were shown to catalyze the oxidation of CO to C02 in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate, or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)+ were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of hydrogen gas; the spectrum of electron acceptors was identical for the three substrates, CO, formate, and H2. The CO- and the formate-oxidizing activities were found to be soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate, and H2 were measured spectrophotometrically following the reduction of methylene blue. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics; the apparent Km for CO was 45 ,uM. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (AH0) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor hydrogen gas is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase, and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide, and fluoride as well as on the failure to trap free formate or hydrogen gas in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans:

CO + H20 -* C02 + 2 H+ + 2e- The CO-oxidizing activity of P. carboxydovorans differed from that of Clostrid- ium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.

The first reports on carbon monoxide oxida- source of carbon and energy (18). Growth on CO tion by aerobic bacteria date back to the begin- was accompanied by the production of carbon ning of this century (2, 11, 17, 27). The early dioxide; 16% of the CO was converted into cell literature was critically reviewed by Kistner (14), carbon as follows (18): who succeeded in isolating a hydrogen bacte- 1 02 + 2.19 CO -0 1.83 C02 + 0.36 cell carbon rium named Hydrogenomonas carboxydovor- + energy ans, capable of oxidizing CO to C02 (15). Since then further aerobic bacteria have been reported Since little is known about the CO oxidation on as the main carbon and reaction proper and since there are no reports to grow CO energy so far on the enzymic mechanism of the conver- source: Seliberia carboxydohydrogena (21, 22, it was of 31), Pseudomonas carboxydoflava (19, 31), sion of CO to C02 in aerobic bacteria, Pseudomonasgazotropha (19,31), Comamonas interest to study this reaction in cell extracts of P. carboxydovorans. The present work was un- compransoris (19, 31), Achromobacter carbox- dertaken to demonstrate the in vitro conversion ydus (19, 31), Pseudomonas carboxydovorans of CO to C02 by crude or partially fractionated (18), the unidentified strains 460 and 461 (D. H. cell extracts of CO-grown P. carboxydovorans Davis, Ph.D. thesis, University of California, some basic of the Berkeley, 1967), and the N2-fixing strains S17 and to elucidate properties and A305 (20). CO-oxidizing system. Pseudomonas carboxydovorans, a gram-neg- MATERIALS AND METHODS ative hydrogen bacterium, has been shown to Chemicals. All chemicals were commercially avail- grow aerobically on carbon monoxide as sole able. 811 812 MEYER AND SCHLEGEL J. BACTERIOL.

Organism and cultivation. P. carboxydovorans is presented in Table 1. A total of 61% of the CO- OM5 (DSM 1227) was grown CO-autotrophically in oxidizing activity and 44% of the formate-oxidiz- mineral medium under the conditions described (18). ing activity were found in the soluble fraction, Cells were cultivated in a 10-liter fermentor (Biostat; but 96% of hydrogenase activity was recovered Braun, Melsungen), harvested in midexponential in the sediment. The percentages of CO-oxidiz- growth phase, washed twice in 50 mM potassium in and the frac- phosphate buffer (pH 7.0), and stored at -20°C. ing activity the soluble particle Preparation of cell extracts. The thawed cells tion turned out to be nearly equal. Because this were resuspended in buffer (2 g [wet weight] of cells in observation suggested the existence of both a about 5 ml of buffer with the addition of 0.4 mg of soluble and a particulate CO-oxidizing activity, DNase) and passed through a French pressure cell at the distribution of enzyme activities was exam- 147.1 MPa (1,500 kpond/cm2) or disrupted by sonic ined by sucrose gradient centrifugation. oscillation (15 s/ml) with a Braun-Sonic 300 disinte- The localization of hydrogenase, CO-oxidizing grator (Quigley-Rochester, Rochester, N.Y.) at 0°C. activity, formate-oxidizing activity, and NADH T'he suspension was centrifuged for 2 h at 100,000 oxidase in CO-grown cells of P. carboxydovor- x g. The supernatant was referred to as soluble frac- ans was studied by fractionation of crude ex- tion. The sediment was clearly separated into two or different layers; the upper layer, which contained the tracts prepared by sonic disruption (Fig. 1A) membranes, was suspended in buffer and designated with a French pressure cell (Fig. 1B) in discon- particle fraction. Mixtures of both fractions were called tinuous sucrose gradients. The almost exact co- crude extract. incidence of activity profiles of hydrogenase and Sucrose gradient centrifugation. Discontinuous NADH oxidase (Fig. 1A and B) unambiguously sucrose gradients were prepared in 13-ml centrifuge disproved the existence of a soluble hydroge- tubes by layering sucrose solutions (2 ml) of different nase. Both the CO- and the formate-oxidizing concentrations (30 to 80%, wt/vol) on top of one an- activities were localized in the 30% sucrose layer, other. Sucrose was dissolved in 50 mM phosphate which contained only a few small particles. buffer (pH 7.0). A 1-ml volume of crude extract was The particles of sonic and French press ex- layered on the top sucrose layer. The tubes were centrifuged at 30,000 rpm for 20 h in a Christ Vacufuge tracts behaved differently during sucrose gra- (Osterode) and fractionated (0.4-ml fractions), and the dient centrifugation, as indicated in the distri- enzyme activities were determined. bution pattern of hydrogenase activity. When Protein determination. The methods of Bradford sonic extracts were used, the hydrogenase activ- (4) or Beisenherz et al. (3) were employed for protein ity profiles exhibited pronounced peaks in the 60 estimation. and 80% sucrose layers (Fig. 1A). In contrast, Methylene blue reduction. The reduction of hydrogenase of French press extracts was bound methylene blue with CO, or formate was measured H2, to smaller and apparently much more homoge- photometrically at 30°C by following the change in neous particles localized in the 50% sucrose layer absorbance at 615 nm (ejI5 = 37.1 cm2/[.mol) using a Zeiss PM6 photometer in connection with a Goerz Servogor recorder. TABLE 1. Distribution of specific CO-, formate-, and Solubility of carbon monoxide and hydrogen. H2-oxidizing activities after centrifugation of a At 30°C and atmospheric pressure, 19.4 yl of CO or crude sonic extract of P. carboxydovorans" 16.7 [i of H2 is dissolved in 1 ml of water (13). Preparation of gas mixtures. Gas mixtures of Oxidizing activity (nmol min-' carbon monoxide, hydrogen, and nitrogen were pre- Substance oxidized mg-') in: pared by filling the required quantities of gas into a syringe (100 ml). CO (99.997 and 99.0 vol%), H2 (99.9 Supernatant Pellet vol%-,), N2 (99.99 vol%), 02 (99.995 vol%), and CO2 CO 99 64 (99.995 vol%) were obtained from Messer Griesheim Formate 91 116 GmbH, Dusseldorf, Germany. H2 41 876 RESULTS 'Assay: 0.91 ml of 50 mM KH2PO4-KOH buffer (pH 7.0); 0.05 ml of 2 mM glucose in buffer; 0.02 ml of 2.5 P. carboxydovorans grew on carbon monoxide mM methylene blue in buffer; 0.01 ml of mix (1 U of (CO-02-N2, 40:5:55) as sole source of carbon and glucose oxidase + 1 U of catalase in buffer); serum- energy (t,1 = 20 h); growth with H2-02-C02 gas stoppered cuvettes were flushed with the following (90:5:5) was much faster (td = 7 h). CO-oxidizing gases for at least 5 min: CO (CO-oxidizing activity), H2 activity was found in CO-grown cells only; hy- (H2-oxidizing activity), N2 in the presence of 0.1 ml of sodium formate (in buffer) in the presence of 0.81 ml drogenase and formate oxidase were also present of 50 mM KH2PO4-KOH buffer (pH 7.0) (formate- under these conditions. oxidizing activity); start with 10 1l of extract (super- Localization of enzyme activities. The dis- natant after 2 h, 100,000 x g) of CO-grown cells of P. tribution of hydrogenase, CO-, and formate-oxi- carboxydovorans and 1 to 8 mg of soluble protein or dizing activities between soluble fraction and the 4 to 14 mg of particle protein per ml; reduction of sediment after centrifugation (2 h, 100,000 x g) methylene blue was measured at 615 nm, 30°C. VOL. 137, 1979 CO OXIDATION IN P. CARBOXYDOVORANS 813 gradient centrifugation, both the CO- and the formate-oxidizing activities must be due to soluble enzymes. Electron acceptors. Crude extracts of CO- grown P. carboxydovorans readily catalyzed the oxidation of carbon monoxide with basic dyes of the thiazine group such as methylene blue or thionine (Lauth's violet) and with dichlorophen- olindophenol, pyocyanine, and toluylene blue (Table 2). Dyes with a potential lower than -100 mV [NAD(P)+, the viologens] were not reduced. Polarographic measurements (Yellow Springs electrode) showed that the reduction of 02 with CO was catalyzed by the particle fraction exclusively, but not by the soluble fraction (2 h, 100,000 x g). For hydrogenase and the formate-

TABLE 2. Reduction of electron acceptors with carbon monoxide catalyzed by the 100,000 x g supernatant of P. carboxydovorans" CO oxidation rates Electron acceptor (mV) ( of rate with methylene blue)5 Carbon monoxide -540 Methyl viologen -440 0 Benzyl viologen -359 0 Neutral red -320 0 NADP+, NAD+ -320 0 FAD+, FMN+c -219 0 Riboflavin -298 0 Indigo (tri-, tetra-)sul- -111 (-70, 0 fonates -30) Vitamin K, -50 3 Vitamin K1 -44 7 Pyocyanine -34 83 Methylene blue +11 100 Thionine (Lauth's vi- +70 30 olet) Phenazine methosul- +80 70 fate Toluylene blue +110 88 0 5 10 1 5 20 25 30 Dichlorophenolindo- +217 20 Fraction number phenol FIG. 1. Distribution patterns of the CO-, formate-, Cytochrome c +245 0 H2-, and NADH-oxidizing enzyme activities of crude Ferricyanide +429 0 extracts of P. carboxydovorans after sucrose density Oxygen +816 0 centrifugation. (A) Sonic extract; (B) French pressure cell extract. After cell disintegration the crude extract "Assay: 1.1 rml of 50 mM KH2PO4-KOH buffer (pH + was layered on top of a 30 to 80% sucrose density 7.0); 0.1 ml of mix (1 U of glucose oxidase 1 U of gradient and centrifuged for 20 h at 30,000 rpm; catalase); 0.5 ml of 2 mM glucose; 0.3 ml of soluble NADH oxidase activity was measured according to fraction (100,000 x g, 2 h, 3.9 mg of soluble protein per Aggag and Schlegel (1). All other enzyme assays are ml) of P. carboxydovorans in the main compartment; described in the legend to Table 1. Symbols: CO (0), 0.2 ml of KOH (20%) in the central cup; 0.3 ml of 7.5 formate (0), H2 (U), and NADH (O). mM electron acceptor in buffer (water-insoluble vita- mins in ethanol); atmosphere 100% CO; start with electron acceptor. with a shoulder in the 60% sucrose layer (Fig. 'CO uptake rates were manometrically measured 1B). From these results the conclusion is drawn at 30°C; the rate with methylene blue was 124 nmol that the P. carboxydovorans hydrogenase is a min-' mg-'. membrane-bound enzyme exclusively. In con- 'FADW, Oxidized flavine adenine dinucleotide; trast, according to results obtained by sucrose FMN+, oxidized riboflavine 5'-phosphate. 814 MEYER AND SCHLEGEL J. BACTERIOL. oxidizing activity an electron acceptor spectrum Methylene blue was reduced by carbon monox- was found identical to that of the CO-oxidizing ide at a 1:1 ratio. This indicates that CO oxida- activity. Therefore, the reduction of methylene tion provides two electrons and carbon dioxide blue followed spectrophotometrically at 615 nm is the reaction product. was used as a standard assay in the following Inactivation by heat. The CO-oxidizing ac- studies. tivity was relatively heat stable (Fig. 4). Portions pH dependence. Maximum methylene blue of 5 ml of the soluble fraction were incubated in reduction rates with CO were found in the range a water bath (80°C); at intervals, samples were of pH 6.5 to 7.2 (Fig. 2). The pH curve shows no taken and cooled in ice. After 2 min of exposure inflection point and is quite different from that to 80°C, the formate-oxidizing activity disap- reported for CO oxidation catalyzed by extracts peared, whereas 40% of the CO-oxidizing activity of Clostridium pasteurianum (24) or Methano- remained. This indicates that the formate-oxi- bacterium thermoautotrophicum (5). Temperature dependence. Maximum rates of methylene blue reduction with CO were found at 65°C (Fig. 3); this value is relatively high for an enzyme functioning in a mesophilic bacterium and may be considered a hint for the involvement of a transition metal. The Arrhenius plot revealed an activation energy (AH0) of 35.9 kJ/mol (8.6 kcal/mol). Stoichiometry of methylene blue reduction with CO. Due to the low apparent Km of 45 [zM CO, the clearly exergonic reduction of methylene blue (E"' = +11 mV) with carbon monoxide (Et') = -540 mV) proceeded to com- >1 plete oxidation of the substrate. Thus the stoichiometry of the reaction could be determined z by adding limiting amounts of CO and by mea- suring the amount of methylene blue reduced. 0 10 20 30 40 50 60 70 80 Temperature (OC) FIG. 3. Temperature dependence ofthe CO-oxidizing activity. Methylene blue reduction as described in the footnote to Table 1. 0o3 -100

-0 E u') 0 .2 (D 50I -0

Z O.1i I 10 0 i t I 0 1 2 3 4 5 6 7 0 Time (min) 3 4 5 6 7 8 FIG. 4. Differentiation of CO-oxidizing activity pH and formate-oxidizing activity of the soluble fraction FIG. 2. pH dependence of the rate of methylene by exposure to 80°C. The soluble fraction of the crude blue reduction with CO. Assay: buffers as indicated; sonic extract (2 h at 100,000 x g) was incubated at methylene blue reduction as described in the footnote 80°C, samples were taken at intervals, and the rate to Table 1. Symbols: (-) KH2PO4-NaOH, 50 mM; of methylene blue reduction was measured with CO (O) Tris-maleate, 50 mM; (A) citrate-Na2HPO4, 100 (-) or formate (0) as described in the footnote to mM. Table 1. VOL. 137, 1979 CO OXIDATION IN P. CARBOXYDOVORANS 815 dizing activity is not involved in the CO oxida- soluble NAD-specific hydrogenase of Alcali- tion reaction. genes eutrophus (Fig. 6). After addition of this Effects of inhibitors. Hypophosphite hydrogenase to the reaction mixture, however, (NaH2PO2), chlorate (NaCl03), cyanide NAD+ reduction was not detectable (Fig. 6, B). (NaCN), and azide (NaN3) are known as metal- Controls performed by the addition of small complexing agents and used as inhibitors of for- amounts of hydrogen-saturated buffer (Fig. 6, C mate dehydrogenases. At high inhibitor concen- and D) or of methylene blue (Fig. 6, E) showed trations, the formate-oxidizing activity as well as that the hydrogenase remained still active in the the CO-oxidizing activity was very low (Table reaction mixture and that the CO-oxidizing ac- 3). However, the formate-oxidizing activity was tivity was still capable of oxidizing CO. subject to a much stronger inhibition than the A similar assay was used to couple the for- CO-oxidizing activity. In the presence of fluoride mation of formate with the reduction of methyl ([NaF] > 300 mM) the CO-oxidizing activity was viologen catalyzed by extracts of Clostridium scarcely affected, whereas the formate-oxidizing cylindrosporum (Fig. 7). However, the dye was activity was almost completely suppressed (Fig. 5). 100 .0 Kinetics of methylene blue reduction p p with CO. The reduction of methylene blue by the soluble fraction of the sonic extract with CO proceeded linearly with time (0 to 3 min), and - the rate was proportional to the amount of pro- 50 tein added in the range measurable photometrically (E615 < 2.5). The dependence of the methylene blue reduction rate on the substrate con- centration (CO) followed simple Michaelis-Men- 10 ten kinetics: plots of 1/v versus 1/[S] were linear. The Km turned out to be 45 ,aM CO. 0 100 [NaF](mM) 500 Exclusion of hydrogen gas and free formate as intermediates of the CO oxidation FIG. 5. Differentiation ofthe CO-oxidizing activity and formate-oxidizing activity of the soluble fraction reaction. Cells of P. carboxydovorans growing by inhibition by fluoride. The rate of methylene blue CO-autotrophically contain hydrogenase and reduction was measured as described in the footnote formate oxidase (18). Thus, the oxidation of to Table 1. carbon monoxide to carbon dioxide and hydrogen gas (CO + H20 -' CO2 + H2; AGO' = -20.1 kJ [-4.8 kcal]) (25) and the hydratation to free formate (CO + H20-- HCOO- + H+; AGO = -16.3 kJ [-3.9 kcal]) (25), which is afterwards oxidized to C02, have to be considered as alter- nate possibilities of CO oxidation; both reactions are thermodynamically possible. If hydrogen gas is formed during the oxidation of CO it should be possible to couple this reaction to the reduction of NAD+ catalyzed by the

TABLE 3. Effect of metal-complexing agents on the CO- and the formate-oxidizing activities" Inhibition (%) 20 Inhibitor Ti,me (min (100 mM) CO Formate FIG. 6. Experiment to detect hydrogen gas as a oxidation oxidation possible intermediate of CO oxidation. Assay: 0.91 ml Hypophosphite (NaH2PO2) 11 89.4 of 50 mM KH2PO4-KOH buffer (pH 7.0); 10 ,ul of mix Chlorate (NaCI03) 0 64.1 (1 U ofglucose oxidase + 1 U of catalase); 50 ILI of 2 Cyanide (NaCN) 64.1 100 mMglucose + 30 LM NAD+; serum-stoppered cuvettes Azide (NaN3) 92.2 100 were flushed with CO. Arrows: A, addition of20 u1 of Fluoride (NaF) 2.3 81.7 crude bacterial extract of P. carboxydouorans; B, 20 fLl of NAD+-specific hydrogenase; C, 100 /1l of H2- " Assay: the CO-oxidizing activity and the formate- saturated buffer; D, 50 u1 of H2-saturated buffer (so oxidizing activity were assayed according to the legend far measured at 365 nm); E, 10I l of2.5 mM methylene to Table 1, but in the presence of 100 mM inhibitor. blue (measured at 615 nm). 816 MEYER AND SCHLEGEL J. BACTERIOL. not reduced; addition of formate d(emonstrated or phenazine methosulfate were readily reduced that the extract was still capable c)f catalyzing by carbon monoxide, whereas oxidized flavine the reduction of methyl viologen w,ith formate. adenine dinucleotide, oxidized riboflavine 5'- The results clearly indicate that neither hydro- phosphate, cytochrome c, and the pyridine nu- gen gas nor free formate is an intermediate of cleotides turned out to be ineffective. This sup- the carbon monoxide oxidation reac tion. ports the assumption that the electrons are de- Other properties of the CO oxidation re- livered at the level of succinate dehydrogenase action. The CO-oxidizing activity w.as only pres- and that a quinone is possibly the physiological ent in CO-grown cells of P. carbo)xydoL'orans electron acceptor. and was very sensitive to alterat.ions of the Centrifugation of extracts in sucrose gradients growth conditions; it was not lost wihen extracts characterized hydrogenase as a membrane- were aerobically dialyzed for 24 h against 50 mM bound enzyme, whereas the CO- and the for- phosphate buffer (pH 7.0), whereas tthe formate- mate-oxidizing activities were soluble or only oxidizing activity was completely destroyed. slightly attached to the membrane; their distri- Ethylenediaminetetraacetic acid (5'0 mM) was bution at a 1:1 ratio between the soluble and without effect on the CO-oxidizing aictivity. The particle fraction after centrifugation may be due reduction of methylene blue with CO started to partial adsorption of the soluble enzyme to immediately after addition of solulble fraction; the particles. however, the reaction mediated by the particle Dehydrogenation of CO is probably due to a fraction sometimes started after a laLg of up to 15 separate enzyme and does not involve formate min. dehydrogenase or hydrogenase activity. This The CO-oxidizing activity was as,sayed under conclusion is based on the differential sensitivity strictly anaerobic conditions, indicating that mo- of the enzyme activities to heat and metal-com- lecular oxygen is not involved and the second plexing inhibitors such as NaF, as well as on the oxygen atom in the CO2 produceci is derived failure to trap free formate or hydrogen gas in from water; thus, CO oxidation is riot due to a coupled optical assays. monooxygenase. The basic reaction actually used in P. carbox- ydou,orans is CO + H2O -* CO2 + 2 Ht + 2e DISCUSSION The reaction serves two different functions: (i) During aerobic growth of P. carbo rydoLtorans to provide electrons for energy generation and with carbon monoxide as the sole source of reductive syntheses and (ii) to produce CO2 for carbon and energy, CO is oxidized to CO2 (18). the reductive pentose phosphate cycle. The CO- CO oxidation was also shown to occutr in extracts oxidizing system thus serves catabolic functions; of CO-grown cells. Different electroin acceptors it is inducible and is only formed during growth such as pyocyanine, methylene bluie, thionine, on CO. Hydrogenase is inducible and is only present in cells grown with hydrogen (H2,-02- CO2) or with pyruvate, whereas the formate- oxidizing activity is constitutive and is present in cells grown with CO (CO-O0), H2 (H2-02- GCO,), acetate, or pyruvate. In contrast, the physiological function of CO- oxidizing activity in methanogenic bacteria (5, 7, 8, 16, 23), sulfate-reducing bacteria (28, 29, 30), photosynthetic bacteria (26), and clostridia (9, 1)0, 24) is far from clear. In these bacteria the A B c c| CO-oxidizing activity is constitutive. It may be due to the nonspecific activities of enzymes ful- filling quite different physiological functions, e.g. in the case of methane monooxygenase (2, 6). 2_____ Furthermore, the CO-oxidizing activities of the Time (min)2 anaerobic bacteria have been shown to reduce FIc. 7. Experiment to detect formate as apossible methylene blue only slightly, but viologen dyes intermiediate of CO oxidation. Assay: c(omponents as with high rates, whereas extracts of P. carbox- in the legend to Fig. 6, but in the pres;ence of 5 [aMW vdo'orans are not capable of oxidizing CO with methyl ciologen instead of NAD+. At-rr 9)VS: A, addi- vriologen dyes as electron acceptors. tion of 10 til of crude bacterial extract cif P. carboxy- docorans; B, 10 pl of craude extract off C. cylindro- ACKNOWLEDGMENTS sporum containing about 0.15 U of foaimq7ntprPltlCt (XUctlY(lh>vr1r)-t - (KOLDINT genase; C. .30 yl of 5 mM formate. We thiank K. Schneider for providing SOIlIble hydrogenase VOL. 137, 1979 CO OXIDATION IN P. CARBOXYDOVORANS 817 of A. eutrophus and R. Wagner for providing extracts of C. 15. Kistner, A. 1954. Conditions determining the oxidation cylindrosporum. of carbon monoxide by Hydrogenomonas carboxydo- Ser. C 57:186-195. CITED vorans. Proc. K. Ned. Akad. Wet. LITERATURE 16. Kluyver, A. J., and C. G. Schnellen. 1947. On the 1. Aggag, M., and H. G. Schlegel. 1973. Studies on a gram- fermentation of carbon monoxide by pure cultures of positive hydrogen bacterium, Nocardia opaca lb. III. methane bacteria. Arch. Biochem. 14:57-70. Purification, stability and some properties of the soluble 17. 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