Carbon Monoxide-Insensitive Respiratory Chain of Pseudomonas Carboxydovorans

JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1178-1187 Vol. 156, No. 3 0021-9193/83/121178-10$02.O0/0 Copyright C 1983, American Society for Microbiology

Carbon Monoxide-Insensitive Respiratory Chain of Pseudomonas carboxydovorans HERIBERT CYPIONKA AND ORTWIN MEYER* Institut fur Mikrobiologie der Universitat Gottingen, D-3400 Gottingen, Federal Republic of Germrany Received 5 July 1983/Accepted 22 September 1983 Experiments employing electron transport inhibitors, room- and low-temperature spectroscopy, and photochemical action spectra have led to a model for the respiratory chain ofPseudomonas carboxydovorans. The chain is branched at the level of b-type cytochromes or ubiquinone. One branch (heterotrophic branch) contained cytochromes b558, c, and a,; the second branch (autotrophic branch) allowed growth in the presence of CO and contained cytochromes b561 and o (b563). Electrons from the oxidation of organic substrates were predominantly channelled into the heterotrophic branch, whereas electrons derived from the oxidation of CO or H2 could use both branches. Tetramethyl-p-phenylenediamine was oxidized via cytochromes c and a exclusively. The heterotrophic branch was sensitive to antimycin A, CO, and micromolar concentrations of cyanide. The autotrophic branch was sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide, insensitive to CO, and inhibited only by millimolar concentrations of cyanide. The functioning of cytochrome a1 as a terminal oxidase was established by photochemical action spectra. Reoxidation experiments established the functioning of cytochrome o as an alternative CO-insensitive terminal oxidase of the autotrophic branch.

A remarkable characteristic of carbon monox- sensitive branch and a novel CO-insensitive ide-oxidizing bacteria, separating them from the cytochrome o (cytochrome b563) functions as an great majority of other aerobic organisms, is alternative terminal oxidase, enabling carboxy- their ability to withstand inhibition by CO (8, dotrophs to withstand CO inhibition. 24). CO insensitivity of carboxydotrophs does not depend on the presence of CO oxidase; MATERIALS AND METHODS therefore, detoxification of CO by the action of Organisms and cultivation. P. carboxydovorans CO oxidase can be excluded (8). OM5 was grown CO autotrophically in 10-liter fermen- Carboxydotrophs have been shown to contain tors (Biostat; Braun, Melsungen, Federal Republic of normal electron transport systems, with b-, c-, Germany) supplied with a gas mixture of (vol/vol) 50%6 and a-type cytochromes at concentrations that CO plus 50%o air as described previously (7, 22). Cells compare to those in other aerobic bacteria (8a). were harvested in the late exponential growth phase and stored at -20°C. CO did not induce the formation of special CO- Preparation of extracts. Cells were disrupted by insensitive terminal oxidases, and the gross means of a French pressure cell at maximum pressure. composition of the respiratory chains was not Whole cells and cell debris were removed by centrifu- affected by the type of growth substrate; CO gation for 10 min at 10,000 x g. Soluble and particulate insensitivity of carboxydotrophs is a constitu- fractions were obtained by centrifugation of cell-free tive property (8). As previously suggested, the extracts for 1 h at 150,000 x g. respiratory chains of Pseudomonas carboxydo- The methods of Bradford (3) and La Riviere vorans (8, 8a) and of Pseudomonas carboxydo- (J. W. M. La Riviere, Ph.D. thesis, University of hydrogena (17, 17a) are branched. Delft, The Netherlands, 1958) were employed for that protein determination. In this communication we demonstrate Oxidase activities. Oxygen uptake of cell extracts branching of the respiratory chain ofP. carboxy- was determined polarographically by means of an dovorans occurs at the level of b-type cyto- oxygen electrode (Yellow Springs Instruments, Yel- chromes or ubiquinone. The electron transport low Springs, Ohio) at 30°C. Experiments were done in system of this bacterium is composed of a CO- 50 mM potassium phosphate buffer (pH 7.0) saturated sensitive and a CO-insensitive branch. Cyto- with a gas mixture of (vol/vol) 50% air and 50o N2. chrome a, is the terminal oxidase of the CO- The reaction was started by injection of the substrates 1178 VOL. 156, 1983 RESPIRATORY CHAIN OF P. CARBOXYDOVORANS 1179 (3 mM NADH, 10 mM succinate, 10 mM formate, or Effect of carbon monoxide on oxidase activities. 10 mM tetramethyl-p-phenylenediamine [TMPD]; the Oxygen uptake of cell-free extracts with CO as latter was reduced with 2 mM ascorbate). H2 oxidase substrate did not depend on the concentrations and CO oxidase were assayed in buffer saturated with of CO (1 to 90% [vol/vol]) and 02 (1 to 15% gas mixtures of (vol/vol) 10%02 and 50o H2 or 10% 02 applied (Fig. 1); this pertained also to and 30 to 90%o CO; N2 served as a balance. CO [vol/vol]) concentrations lower than 30% (vol/vol) were obtained the succinate and formate oxidase activities. by adding CO-saturated buffer. Oxygen uptake with these substrates in the The inhibitors rotenone, thenoyltrifluoroacetone presence of CO was increased by the amount of (TTFA), antimycin A, dicumarol, and oligomycin oxygen uptake with CO alone. However, when were dissolved in ethanol; 2-n-heptyl-4-hydroxyquino- CO oxidase was specifically inhibited by 2% line-N-oxide (HQNO) was dissolved in dimethyl forrh- (vol/vol) methanol (21), the same rates as in the amide. All reaction rates of oxidases were corrected absence of CO were obtained. Oxidase activities for solvent effects. with NADH and H2 were sensitive to CO (Fig. Spectrophotometric techniques. Difference spectra partially inhibited were recorded at liquid nitrogen or room temperature 1). Low CO concentrations on a Hitachi model 556 spectrophotometer (Hitachi, NADH oxidase and H2-oxidase by 25 and 13%, Tokyo, Japan) as' described previously (8a). Absorp- respectively, but at CO concentrations above tion peaks of the cytochromes at low tenmperature 20% (vol/vol), CO inhibition did not increase showed a blue shift of 2 to 3 nm; therefore, the (Fig. 1). These results are indicative of the subscripts LT (low temperature) or RT (room tem- presence of two terminal oxidases, one of them perature) are used to refer to liquid nitrogen or room being insensitive to CO. temperature spectra, respectively. Photochemical CO TMPD oxidase was very CO sensitive; at low action spectra were recorded with the oxygen elec- CO saturation the oxygen uptake in the presence trode placed in a dark chamber, provided with an mM TMPD was inhibited by 42%, and aperture that allowed illumination of the assay. Light of 10 of different wavelengths was obtained with a super inhibition increased to about 60% at high CO pressure mercury lamp (Osram; HBO 200) and preci- levels (Fig. 1). sion monochromatic filters of transmission half-widths of 10 nm (type DIL; Schott, Mainz, Federal Republic of Germany). Light intensity was determined with a luxmeter (Metrux K; Metrawatt GmbH, Nurmberg, Federal Republic of Germany). Action spectra were recorded at 30% (vol/vol) CO saturation. Chemicals. NADH, antimycin A, oligomycin, ADP, EEn 100 ATP, catalase, and superoxide dismutase were from Boehringer, Mannheim, Federal Republic of Germa- a ny; Dicumarol, rotenone, HQNO, TTFA, and TMPD .E were from Sigma Chemical Co., St. Louis, Mo. All other chemicals were from Merck AG, Darmstadt, N S. - Federal Republic of Germany. 0 'U 0 -~~~4,...--- -, E RESULTS S 50 au Oxidase activities in cell extracts. The specific oxidase activities of CO-grown P. carboxydo- I I I I I I I -I vorans with NADH, succinate, or TMPD were 0. vII 1.3 to 2 times higher in the particle fraction than m in cell-free extracts, whereas the highest oxidase N activities with formate or CO were found in cell- free extracts. This is in accordance with the 0 distribution of formate oxidase and CO oxidase 60 80 (23). No oxidase activities with NADH, succi- 0 20 40 nate, H2, or CO occurred in the supernatant CO Saturation (%) fraction, and the activities with formate or TMPD were only minor. No effect on the oxi- FIG. 1. Influence of CO on oxidase activities. Oxy- addition of gen uptake was measured polarographically with cell- dase activities was observed upon (i) free extracts of CO-grown P. carboxydovorans at 10% 1 FLM to 1 mM dinitrophenol or dicumarol as oxygen saturation. Methanol (2% (vol/vol]) was added uncouplers, (ii) 1 to 100 ,ug of oligomycin per ml to inhibit CO oxidase, except when CO served as a as an inhibitor of energy-linked electron trans- substrate. The following substrates were used: H2 (0), port, or (iii) 0.2 mM ADP or ATP; this is NADH (U), CO (0), succinate (O), formate (A), and indicative of uncoupling of respiration and phos- TMPD (A). The TMPD oxidation rates indicated are phorylation in the membrane preparations used. divided by a factor of 10. 1180 CYPIONKA AND MEYER J. BACTERIOL. Effect of light on CO inhibition. The CO- insensitive oxidase activities with succinate, formate, or CO of cell-free extracts were not influ- 1.0 enced by irradiation with white light, regardless of the presence or absence of CO (Table 1). a However, the partial inhibition of NADH oxidase and H2 oxidase by CO was almost completely abolished upon exposure to light. TMPD 4 oxidase activity measured in the dark was re- 0 50 duced by CO to 35%, but increased about twice 0DI c when light was on (Table 1). The effect oflight of 0E different wavelengths on inhibition of TMPD 4- _ oxidase by CO was measured (Fig. 2) to obtain ._ the photochemical CO action spectrum (5, 6). The maximum at 595 nm confirmed the role of I cytochrome a as the CO-sensitive terminal oxi- °t I I I I I dase. The absence of relief of CO inhibition by 4CK0 50 Bo 50 600 660 light of 626 or 649 nm argues against the pres- Wavelength (nm) ence of a functioning cytochrome d (a2). The FIG. 2. Photochemical CO action spectrum of the maximum of light relief was at 422 nm, and only TMPD oxidase of P. carboxydovorans. The spectrum 55 lux oflight ofa 422-nm wavelength caused the was obtained as described in the text. The intensities same grade of relief from CO inhibition as did of the light applied were 450 lux at wavelengths of 499 white light of 60,000 lux (Table 1). From the to 649 nm and 55 lux at wavelengths of 408 to 441 nm. peak at 596 nm in difference spectra and from the peaks at 422 and 595 nm in its action HQNO caused only partial inhibition (less than spectrum the CO-sensitive terminal oxidase of 30%). However, titration of that enzyme with P. carboxydovorans may be assigned as cyto- KCN and NaN3 revealed biphasic curves. This chrome a1. finding points to the presence of two different Effect of electron transport inhibitors on oxi- terminal oxidases with different affinities for dase activities. The respiratory chain of CO- these two inhibitors. grown P. carboxydovorans was studied with the Succinate oxidase was also inhibited by all of electron transport inhibitors rotenone (1, 11, 13), the electron transport inhibitors mentioned, but TTFA (30), antimycin A (1, 2, 12), HQNO (13, inhibition was presumably unspecific with rote- 19), KCN, and NaN3 in the presence of different none, as indicated by the high Ki of 1.3 mM substrates as electron donors. (Table 2). As with NADH, HQNO caused only NADH oxidase was affected by all inhibitors partial inhibition of succinate oxidation. The mentioned (Table 2 and Fig. 3a). Titration sensitivity of succinate oxidase to antimycin A curves of NADH oxidase with rotenone, TTFA, (Ki = 0.69 mM) was 20-fold lower compared to antimycin A, and HQNO were monophasic, and NADH oxidase. All inhibition curves were monophasic, including those with KCN or NaN3. TABLE 1. Influence of light on the oxidase Formate oxidase was insensitive to rotenone, activities in cell-free extracts in the presence of CO antimycin A, and HQNO. Sensitivity to TTFA and NaN3 was ofthe same order ofmagnitude as Oxygen Oxygen uptake' in with NADH or succinate (Table 2). Only for- uptakea in the the presence of mate oxidation was very sensitive to KCN = Substrate absence of 30to CO Stimulation (Ki CO, dark or by light(% 3 ,IM). illuminated' Dark Illuminated' Oxidation of both H2 and CO was inhibited by same NADHC 60.9 48.2 56.8 18 the type of inhibitors and was insensitive Succinatec 12.4 17.1 17.1 0 to rotenone and TTFA (Fig. 3b). Increasing Formatec 7.9 11.0 11.0 0 concentrations of HQNO resulted in a propor- H2c 31.9 27.8 30.8 11 tional decrease of CO-oxidizing activity (Ki = 1 CO 0 15.3 15.3 0 mM); the H2 oxidase was very sensitive to TMPD 686 240 500 108 HQNO (Ki = 5.6 ,uM). Sensitivity to antimycin was = = a The reaction rates are in nanomoles of oxygen per A low with H2 (Ki 3 mM) or CO (Ki 0.7 minute per milligram of protein. mM) compared to NADH oxidase (Ki = 34 FM). b White light was applied at an intensity of 60,000 Titration curves with H2 or CO as substrates lux. with cyanide at different concentrations were c CO oxidase was inhibited by 2% (vol/vol) metha- biphasic (Fig. 3b). nol. TMPD oxidase was inhibited only by KCN VOL. 156, 1983 RESPIRATORY CHAIN OF P. CARBOXYDOVORANS 1181 TABLE 2. Inhibition constants (Kg) for the different electron transport inhibitors and substrates Ki Substratea Rote- 1TFA Antimycin HQNO NaN3 KCN none A NADH 3.5 pLM 4.0 mM 34 ,uM 0.56 mM 6.3 mM 65 ,uM 21 mM 1.8 mM Succinate 1.3 mM 2.8 mM 0.69 mM 4.0 mMb 63 mM 3.1 mM Formate NlV 1.3 mM NI NI 4.3 mM 3.0 ,uM H2 NI NI 3.0 mM 5.6 ,uM 160 mM 0.71 mM 12 mM CO NI NI 0.71 mM 1.0 mM 54 mM 32 ,M 4.0 mM TMPD NI NI NI NI 1.6 mM 14 ,M a Experiments were performed with cell extracts of CO-grown cells of P. carboxydovorans after 2 min of preincubation with the different inhibitors. bInhibition was less than 30%. c NI, No inhibition occurred at an inhibitor concentration of 1 mM. and NaN3 (Table 2), and the titration curves against oxidized extracts in the steady state. with both inhibitors were monophasic. These spectra were recorded with low extract The presence of two different terminal oxi- concentrations. Control experiments with the dases is evident from the occurrence of biphasic oxygen electrode showed that it took more than titration curves with CO, KCN, and NaN3. 5 min to use up all the oxygen present in the Antimycin A was very useful in distinguishing assay, thus allowing measurements within this between the different electron transport routes period of time. All cytochromes remained oxi- actually used by the different oxidases. The dized to 85 to 95% under these conditions. oxidation of NADH was 20- to 30-fold more However, the b-type cytochromes behaved dif- sensitive to antimycin A than was the oxidation ferently; they were reduced to 6% with NADH, of succinate, H2, or CO (Table 2). On the other succinate, formate, or TMPD as substrate and to hand, the oxidation of NADH of succinate was 11% when CO or H2 served as substrate. only partially inhibited by HQNO, whereas the Successive reduction of the cytochromes was oxidation of H2 or CO was very sensitive. This followed by recording difference spectra in short indicated that NADH, on the one hand, and CO time intervals, immediately after the addition of and H2, on the other hand, were oxidized via substrate. With all substrates cytochromes c and distinct branches of the respiratory chain. a were the first to be reduced, followed by the b- It has been reported that formate inhibits type cytochromes. With NADH as substrate it terminal oxidases (27), e.g., cytochrome o of took 2 min to use up all the oxygen present in the Rhodospirillum rubrum or Thiocapsa roseoper- cuvette. At that point cytochromes c and a were sicina (14). However, 100 mM formate had no reduced to 80 and 90%, respectively. In the effect on the oxidation of CO, H2, NADH, or same experiment the cytochrome b pool was succinate with oxygen. reduced to only 18% after 2 min and to 38% after It is also known that the mitochondria of 8 min. certain plants, protozoa, yeasts, and fungi con- Cytochromes c and a were reduced to 82 to tain a cyanide-insensitive electron transport sys- 85% and 100%, respectively, upon incubation tem (for a review, see reference 9). The cyanide- for 1 h with an excess of each of the different insensitive alternative oxidase of these substrates (Table 3). Reduction of the b-type organisms is specifically inhibited by salicylhy- cytochromes depended on the type of substrate. droxamic acid (29). The oxidation of CO, H2, or CO or H2 was able to reduce cytochrome b NADH in cell-free extracts of P. carboxydovor- better than all other substrates; however, com- ans was only slightly affected by salicylhydroxa- plete reduction was never achieved. The appar- mic acid (1 to 20 mM), and the inhibition of these ent reduction of the b-type cytochromes after 1 h reactions by antimycin A or cyanide was not of incubation with CO was 45%. This value further increased by salicylhydroxamic acid. amounted to 74% after correction for binding of This indicates that the CO insensitivity of P. CO to a portion of the b-type cytochromes. carboxydovorans is due to a different type of Low-temperature spectroscopy with different alternative terminal oxidase. electron donors was used to distinguish and Spectroscopic examination of electron trans- further characterize the three b-type cyto- port. Difference spectra with cell-free extracts chromes present in CO-grown P. carboxydovor- were recorded during the oxidation of substrates ans (8a). The dithionite-reduced minus oxidized 1182 CYPIONKA AND MEYER J. BACTERIOL. difference spectrum had its maximum at 558 nm, a peak at 561 nrh, but no band at 556 nm (Fig. 4d). This indicated that cytochrome b561(LT) and particularly b55s8(LT) were more efficiently reduced by ti2 than by NADH, whereas cytochrome b556LT) was reduced to an equal degree by both substrates. These results suggest that cytochrome b5s5LT) is located at the branching point of the respiratory chain or in the NADH branch and that ._- cytochromes b558(LT) and b561(LT) are located in exclusively. A trough at 563 4- the H2/CO branch Li nm (RT) in CO difference spectra corresponding to cytochrome b561(LT) is suggestive of the presence of cytochrome o with terrhinal oxidase function. Inhibitor concentration (M) Effect of electron transport inhibitors on the redox state of cytochromes. The addition of rote- 1. none, TTFA, or HQNO uniformly delayed the reduction of all cytochromes when ,substrates were used whose oxidation was affected by these inhibitors. This indicated that the inhibi- 0 tion sites of these inhibitors were localized before the transition of electrons to cytochromes. Oxi4ation of 112 or CO with methylene blue instead of oxygen as an electron acceptor was not affected by 250 ,uM HQNO; this suggests inhibition by HQNO at the level of ubiquinone. >4 Antimycin A inhibited the transfer of elec- trops to cytochromes c and a. In the absence of inhibitors cytochromes c and a were always reduced before the b-type cytochromes; in the 0 10-5 lo4 1Q3 102 101 presence of antimycin A, however, all three b- Inhibitor concentration ( M type cytochromes were reduced to a higher degr e than cytochromes c and a (Fig. 5a). FIG. 3. Titration with electron transport inhibitor Inhibition of cytochrome a in cell-free ex- of NADH- (a) and CO-oxidizing (b) activities. Oxygen tracts with 5 mM KCN resulted in reduction of uptake was measured polarographically after preincubation of cell-free extracts ofCO-grown P. carboxydo- cytochromes c and a. In the absence of added vorans for 2 min with the following inhibitors: rote- substrate or in the presence of substrates during none (A), antimycin A (m), TTFA (L), HQNO (A), the steady state, only the b-type cytochromes KCN (0), and NaN3 (0). remained cotnpletely oxidized (Fig. 5b). These results indicate that, when cytochrome a is

TABLE 3. Reduction of cytochromes with different difference spectrum revealed two similar peaks substrates at 561 and 558 nm and a shoulder at 556 nm (Fig. Reduction' of cytochrome (%) 4a). The peak at 550 nm originated from cyto- Substrate chrome c. The dithionite-reduced minus H2- a bb c reduced difference spectrum showed a maxi- NADH 100 40 85 mum at 561 nm, a shoulder at 556 nmn, but no Succinate 100 43 83 band at 558 nm (Fig. 4b). This indicated that Formate 100 43 84 cytochrome b558(LT) was completely reduced by H2 100 63 85 H2, whereas cytochromes b5565LT) and b561(LT) CO 100 74C 83 remained partially oxidizrd, the latter even to a TMPD 100 25 82 larger extent than the former. The dithionite- a Room-temperature difference spectra were re- reduced minus NADH-reduced difference spec- corded after 1 h incubation of cell-free extract with an trum also had its maximum at 561 nm (Fig. 4c). excess of substrate. Values given are percentages of The shoulder at 558 nm was indicative of incom- the reduction obtained with dithionite. plete reduction of cytochrome b558(LT) by b Total content of the different b-type cytochromes. NADH. The H2-reduced minus NADH-reduced c See text. VOL. 156, 1983 RESPIRATORY CHAIN OF P. CARBOXYDOVORANS 1183 Complete reoxidation of cytochromes a and c 77KK61 then proceeded concomitantly. Quite similar re- S5b) ^ a) 0.020.0k. oxidation spectra were obtained with succinate, 556 c) 0.02 formate, H2, and CO as reducing substrates. 561 d) 0.01 With TMPD, reoxidation of cytochrome a was 556 observed, but the redox state of the b-type cytochromes remained unchanged. 556 j 556 5501 The presence of 1 mM antimycin A had no effect on the reoxidation spectra. Cyanide (1 mM KCN) blocked the reoxidation of cyto- 1561 chrome a but not of the b-type cytochromes. Reoxidation of all cytochromes completely failed in the presence of 10 mM KCN, even at I 561 o @ ' high oxygen partial pressure (200 air bubbles). These results indicate an electron flow from the b-type cytochromes to oxygen and the functioning of one of the b-type cytochromes as a terminal oxidase. a) b) ~~~~~c)d) 77 K -550 550 560 550 560 550 56 sWuw Wavltength (nm) FIG. 4. Low-temperature difference spectra of membrane vesicles of CO-grown P. carboxydovorans. Spectra were recorded after incubation with the sub- LaA strates for 30 min. The final protein content was 5.7 mg/ml. (a) Dithionite-reduced minus air-oxidized; (b) dithionite-reduced minus H2-reduced; (c) dithionite- reduced minus NADH-reduced; and (d) H2-reduced minus NADH-reduced. blocked by cyanide, the flow of electrons to oxygen proceeds via b-type cytochromes exclusively, but not via cytochrome c. The data are indicative of branching of the respiratory chain 596 at the level of b-type cytochromes. ;60 Reoxidation of reduced cytochromes. More de- tailed information on the terminal oxidases was obtained from reoxidation spectra with cell-free b) extracts of P. carboxydovorans. These spectra were recorded as follows. The baseline was obtained from the difference spectrum of extracts reduced with an excess of substrate; then a) the reference cuvette was reoxidized to an increasing degree with air bubbles. This method produced reoxidation spectra showing the reoxi- a IL L- dized cytochromes in their reduced form. I Such a sequence of reoxidation spectra ob- 50O 550 600 650 tained with NADH-reduced cell-free extract is Wavelngth (n m) shown in Fig. 6. Very small amounts of oxygen FIG. 5. Effect of electron transport inhibitors on (three air bubbles) were sufficient to reoxidize cytochrome reduction. Low-temperature difference 40% of the b-type cytochromes, indicating their spectra were recorded with cell-free extracts of CO- high affinity for oxygen. A plateau at 560 to 563 grown P. carboxydovorans. The protein content was 19.1 mg/ml. (a) Assays contained 1 mM antimycin A, nm and a weak shoulder at 558 nm indicated that NADH-reduced minus air-oxidized after incubation all three b-type cytochromes were reoxidized. A with antimycin A for 5 min; (b) difference spectrum of slight peak at 597 nm was indicative of partial oxidized assays in the presence and absence of 5 mM reoxidation of cytochrome a. Cytochrome c KCN; no electron donor was added, and spectra were remained completely reduced until high oxygen recorded after 15 min. Similar spectra were obtained concentrations were applied (200 air bubbles). during steady-state oxidation of substrates. 1184 CYPIONKA AND MEYER J. BACTERIOL. be expected under these conditions if the latter is a respiratory end product. Upon addition of high concentrations of catalase in the presence or absence of cyanide and immediately after all oxygen in the assay was consumed, no accumulation of H202 was observed under the conditions described by Jensen (15). These data clearly exclude H202 as a major end product of the autotrophic branch of the respiratory chain of P. carboxydovorans. DISCUSSION The data presented in this report clearly establish the presence of both a CO-sensitive and a CO-insensitive electron transport system in P. carboxydovorans and provide an explanation for the unique resistance of carboxydotrophic bacteria toward CO (8). Our results indicate that NADH and H2 or CO are oxidized via different branches of the respiratory chain. (i) NADH

400 450 500 550 600 650 Wavelength (nm) FIG. 6. Successive reoxidation of reduced cytochromes. Two assays containing cell-free extracts of CO-grown P. carboxydovorans (4.5 mg. of protein per ml) were reduced with 3 mM NADH. The reference cuvette was successively reoxidized with air (3 to 800 bubbles), and room-temperature difference spectra were recorded within 1 min.

The time dependence of the absorbance differences between 558, 561, and 563 nm during reoxidation of cell extracts was followed to elucidate the reaction sequence of b-type cytochromes (Fig. 7); absorbancies decreased first at 563 nm, indicating that cytochrome b563 was the first to be reoxidized. This establishes the functioning of cytochrome b563 as a terminal oxidase. Catalase activity and H202 formation. The 0 30 occurrence of a branched respiratory chain with Time Is ) one branch forming H202 rather than water from FIG. 7. Reoxidation of cytochrome b followed by oxygen is known from different organisms and dual wavelength scanning. Cell-free extracts of CO- from mitochondria (20). Therefore, we looked grown P. carboxydovorans (2.1 mg of protein per ml) for H202 formation as the end product of the were incubated with 3 mM NADH for 20 min. To CO-insensitive branch. prevent reoxidation of cytochromes c and a, the The endogenous catalase activity of the cell assays were incubated with 1 mM KCN for an addi- extract (270 nmol of 02 produced per min per mg tional 10 min. At the time indicated by the arrows, the of protein with 0.1 ,uM H202) was reduced to 43 assays were flushed with 200 Ill of air. The absorption differences at (wavelengths in nanometers) 562 minus nmol/min per mg ofprotein by the addition of0.2 552 (a), 562 minus 558 (b), 561 minus 563 (c), and 563 mM KCN; under these conditions the H2 oxi- minus 561 (d) were scanned with an Aminco DW2 dase activity decreased only from 138 to 106 spectrophotometer at a slit width of 0.5 nm. The data nmol of 02 consumed per min per mg of protein. were transferred to a Midan T data analyzer and These data show that accumulation of H202 can smoothed (mode 25) before plotting. VOL. 156, 1983 RESPIRATORY CHAIN OF P. CARBOXYDOVORANS 1185 oxidation was 20- to 90-fold more sensitive to exclusively. As the autotrophic branch was in- antimycin A (Ki = 34 ,uM) that the oxidation of sensitive to low concentrations of antimycin A CO (Ki = 0.71 mM) or H2 (Ki = 3 mM). (ii) and as the heterotrophic branch was only par- Oxidation of CO or H2 was strongly sensitive to tially inhibited by HQNO, branching occurs at HQNO, whereas NADH oxidation was only or just before the sites of action of these inhibi- partially inhibited. (iii) During steady-state oxi- tors. Branching of the electron transport system dation of CO or H2 the b-type cytochromes were at the level of cytochrome c has been suspected reduced to a greater extent than during oxidation for P. carboxydohydrogena (17); that it occurs in of all other substrates. (iv) Even after long P. carboxydovorans at the level of b-type cyto- incubation with CO or H2 the b-type cytochromes or ubiquinone is evident from the fol- chromes were more reduced than after incuba- lowing arguments. (i) The inhibition site of anti- tion with other substrates. Hence we conclude mycin A is located before cytochrome c. (ii) that the b-type cytochromes age preferentially TMPD delivers electrons at the level of cyto- used by electrons that stem from CO or H2 chrome c; its oxidation was very sensitive to CO (autotrophic branch), whereas NADH is prefer- or cyanide and revealed monophasic titration entially oxidized via cytochromes c and a (het- curves. Thus, only a single terminal oxidase erotrophic branch). The fact that electrons from could be used by electrons derived from oxida- CO or H2 could also reduce cytochromes c and a tion of cytochrome c. (iii) The presence of indicates a link between both branches of the cyanide resulted in reduction of cytochromes c respiratory chain. Sensitivity to antimycin A and and a, but the b-type cytochromes remained partial inhibition by HQNO are suggestive of oxidized. This is indicative of electron flow from succinate oxidation via the heterotrophic the b-type cytochromes via the less cyanide- branch. sensitive terminal oxidase to oxygen and dis- The low formate-oxidizing activity and its proves any electron flow from cytochrome c to insensitivity to antimycin A and HQNO made it that terminal oxidase. Inhibition by HQNO of impossible to decide which branch of the respi- the autotrophic branch exclusively may arise ratory chain is preferentially used. The high from a functioning ubiquinone cycle (25, 26) at sensitivity of formate oxidation to cyanide is the branching site. supposedly due to the sensitivity of formate The functioning of cytochrome a as the termi- oxidase to that inhibitor (23) and not to action of nal oxidase of the heterotrophic branch is evi- cyanide on the terminal oxidase. As formate dent from the photochemical CO action spec- oxidation was insensitive to CO and as all cyto- trum of the TMPD oxidase; the former was very chromes were at least partially reduced, this similar to cytochrome a, ofAzotobacter vinelan- substrate can probably be oxidized via both dii (10) and may be assigned to cytochrome a, branches of the respiratory chain. TMPD oxi- from the peaks at 422 and 595 nm. Because of its dase was sensitive to the classic inhibitors of insensitivity to CO, the nature of the terminal terminal oxidases (CO, cyanide, and azide) ex- oxidase of the autotrophic branch could not be clusively. The b-type cytochromes remained ox- examined by use of action spectra. However, idized during steady-state oxidation of TMPD, the following arguments establish cytochrome and only low amounts of them were reduced b563 as a cytochrome o functioning as a CO- during long incubation with TMPD. These data insensitive terminal oxidase of the autotrophic indicate that TMPD is oxidized via cytochromes branch of the respiratory chain of P. carboxydo- c and a exclusively. We conclude from the low- vorans. (i) H202 was not the end product of the temperature spectra with NADH or H2 that autotrophic branch. (ii) Cytochrome d (a2) and cytochromes b558(LT) (b561(RT)) and b561(LT) the alternative terminal oxidase present in many (b563(RT)) are constituents of the autotrophic eucaryotes (9) were absent in P. carboxydovor- branch and that cytochrome b556(LT) (b558(RT)) is ans. (iii) CO difference spectra revealed a maxi- located in the heterotrophic branch or at the mum at 416 nm and troughs at 433 and 563 nm, branching point of the respiratory chain. indicative of cytochrome o (6). (iv) Cytochrome The following data are indicative of a b563 was the first to react with oxygen during branched electron transport system. (i) Oxida- reoxidation of reduced extracts; it was the least tion of NADH or H2 was only partially inhibited reducible cytochrome, even with CO or H2. by CO. (ii) The biphasic titration curves with Cytochrome o is the most important terminal cyanide indicate that two terminal oxidases oxidase found in bacterial respiratory systems, could be reduced by electrons from one single and biochemical and spectral characteristics substrate. (iii) The different substrates tested suggest the existence of several classes (16). could reduce all cytochromes, at least to a Usually cytochrome o is sensitive to CO and can certain degree. That the branching point is locat- be detected by CO action spectra. However, ed after the action site of rotenone is indicated according to Castor and Chance (6), the nonap- by inhibition of NADH oxidase by rotenone pearance of a pigment in CO action spectra does 1186 CYPIONKA AND MEYER J. BACTERIOL. not preclude the possibility of its having oxidase none in P. carboxydovorans described in this activity. Besides, o-type cytochromes exhibiting report. The electron transport system of P. low or even no affinity for CO are known (4, 18, carboxydovorans (Fig. 8) shows noteworthy 30a, 33; W. Tamura-Lis and D. A. Webster, similarities to that of Paracoccus denitrificans Fed. Proc. 41:749, 1982). The cytochrome o of (28, 31, 32). The b-type cytochromes were more P. carboxydovorans was also found to be insen- effectively reduced by H2 than by NADH in CO- sitive to CO under physiological conditions; its grown P. carboxydovorans as well as in H2- existence and functioning as an alternative ter- grown P. denitrificans. Antimycin A blocked minal oxidase provides the clue for the unique only the reduction of the cytochromes c and a of CO resistance of P. carboxydovorans and the P. denitrificans, and cytochrome o had a lower other carboxydotrophic bacteria. The CO-bind- affinity for cyanide than cytochrome a (28). ing spectrum under strictly reduced conditions These considerations agree with our finding that indicates that this cytochrome can indeed react respiration of whole cells of P. denitrificans is with CO. It is reasonable to assume that high insensitive to CO (8) and with the absence of CO affinity for oxygen and relative low oxidase rates action spectra of cytochrome o in extracts of P. keep cytochrome o oxidized and thus prevent denitriJicans (30a). reaction with CO in vivo. The reactions of cytochromes described here A major conclusion from this work is that the for CO-grown P. carboxydovorans, i.e., inhibi- electron transport chain of P. carboxydovorans tion, reoxidation, and sensitivity to CO or cya- is branched into an ordinary CO-sensitive, het- nide, were also examined in extracts of cells erotrophic branch and a CO-insensitive autotro- grown organotrophically with pyruvate or auto- phic branch. Branching occurs at the level of b- trophically with H2 plus CO2 and were found to type cytochromes or ubiquinone, and CO be identical to those described here for CO- insensitivity is due to the presence of a novel grown cells. This confirms that the CO-insensi- CO-insensitive cytochrome o that functions as tive branch of P. carboxydovorans is constitu- an alternative terminal oxidase. The proposed tive. electron transport system of P. carboxydovor- ACKNOWLEDGMENTS ans is depicted in Fig. 8. It agrees with the data of Kim and Hegeman (17, 17a) for P. carboxydo- The stimulating interest of H. G. Schlegel and a grant from the Deuitsche Forschungsgemeinschaft are gratefully acknowl- hydrogena insofar as they suppose a branched edged. We wish to thank G. D. Hegeman and Y. M. Kim for electron transport system with cytochrome o as generously sharing unpublished findings with us. one terminal oxidase. However, the model proposed for the electron transport system in P. LITERATURE CITED carboxydohydrogena assumes branching at the 1. Asano, A., and F. Brodie. 1965. 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