Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas Carboxydovorans
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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 car- boxydovorans 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 ap- parent 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, hydroge- nase, 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 sol- uble 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).