Oxidative Phosphorylation Coupled with Nitrate Respiration

Transcription

1 The Journal of Biochemistry, Vol. 55, No. 2, 1964 Oxidative Phosphorylation Coupled with Nitrate Respiration II. Phosphorylation Coupled with Anaerobic Nitrate Reduction in a Cell-Free Extract of Escherichia coli By AKIHIRO OTA, TATEO YAMANAKA and KAZUO OKUNUKI (From the Department of Biology, Faculty of Science, University of Osaka, Osaka) (Received for publication, August 30, 1963) As is well known, Escherichia coli can grow anaerobically if nitrate is provided. Under these conditions, nitrate is used by this or ganism as an ultimate electron acceptor just like molecular oxygen is in aerobic condi tions. This phenomenon is called nitrate re spiration (1). Indeed, it has been demon strated in cell-free extracts of Pseudomonas aeruginosa (2) and P. denitrificans (3) that ox idative phosphorylation coupled with nitrate respiration occurs. It is of interest to test whether this oxidative phosphorylation cou pled with nitrate respiration also occurs in denitrifying bacteria other than pseudomo nads. Herseyand Aj1 (4) found that oxida tive phosphorylation coupled with aerobic oxidation of succinate occurs in cell-free ex tracts of E. coli. In this case, it was found that ferricyanide could replace oxygen as the oxi dant. However, the occurrence of oxidative phosphorylation coupled with anaerobic re duction of nitrate has not been established. Therefore, in the present investigation, this was studied using cell-free extracts of E, coli. MATERIALS AND METHODS Organism-E. coli (strain 0-5 from the Public Health Research Institute of Kobe City) was grown on bouillon-peptone-agar containing 2% KNO3 in Roux flasks for 18 hours at 37 C. Preparation of Cell-Free Extracts-The cells were harvested and washed three times with 0.1 M KCI containing M Tris buffer (ph 7.4) and then resuspended in the same medium. The suspension was treated in a sonic oscillator (9 kc, 50 W) at about 3 C. This sonic treatment was applied for 30 second period at 30 second intervals for a total sonication neriod of 3 minutes. Then the resulting suspension 131 was centrifuged at 4,000 ~g for 10 minutes. The supernatant was named S4. The supernatant obtained by centrifugation of S4 for 10 minutes at 8,000?g was named S8. Further fractionation was carried out as follows; S8 was centrifuged at 144,000?g for 1 hour and the precipitate obtained was washed once with 0.1 M KCl. After recentrifugation this fraction was named P The supernatant obtained after centri fugation at 144,000 ~g was designated as S144. Reaction Procedures-Nitrate reduction and phosph orylation were measured as described previously (2). Thus, nitrate reduction was carried out anaerobically in Thunberg tubes, and the reaction measured either as nitrate consumed or nitrite formed. Phosphoryla tion was determined using P;32 according to the met hod of Hagihara and Lardy (5). RESULTS As Table I shows, a definite increase in phosphorylation occurred on addition of nitrate under anaerobic conditions with citrate as an electron donor. There was a considerable incorporation of Pill even in the absence of citrate under anaerobic con ditions, and this incorporation was apprecia bly decreased after dialysis of the preparation against 0.1 M KCl for 2 hours. The preparation reduced nitrate to nitrite under the anaerobic conditions, but could not reduce the nitrite further, as shown in Table I. Since 10 Đmoles of KNO3 was add ed to each tube, it is evident from Exp. 2 that all the KNO3 reduced was recovered as KNO2. Thus, the phosphorylation observed here was coupled only with the reduction of nitrate to nitrite. As shown in Fig. 1, the amount of Pi incorporation coupled with anaerobic nitrate reduction reached a con-

2 132 A. OTA, T. YAMANAKA and K. OKUNUKI Phosphorylation Coupled with Nitrate reduction in S4 I The reaction mixture was composed of 0. 1 ml. of hexokinase [EC ] (5 mg./ml.), 0. 1 ml. of 0. 1 M pi32 buffer (approx. 3 X 104 c.p.m./Đmole), 0.1 ml. Of 0. 1 M MgCI2, 0.1 ml. Of 0.1 M glucose, 0.1 ml. of 10-3 M ATP, 0.1 ml. of 0.1 M citrate, 0.I ml. of 0.1 M KNO3, and 0.2 ml. of cell-free extract (5 mg. of protein/0.2 ml.). The total volume was made up to 1.0 ml. with distilled water. The reactions were car ried out anaerobically at ph 7.4 and at 30 C, for 30 minutes in Thunberg tubes. Almost the same re sults were obtained with S6 as with S4. FIG. 1. The time course of phosphorylation with fraction S4. Reaction conditions were the same as for Table I. A; Phosphorylation in the presence of KNO3, B; P/NO3-, C; phosphoryla tion in the absence of KNO3. Fraction S4 con tained 16.5 mg. of protein per ml. stant level 30 minutes after the beginning of the reaction. The P/NO3- ratio (the ratio of Pi incorporated to nitrate consumed) decreased with time during the reaction period. The solution which had been passed through a siliconized Celite column to determine P;3- incorporation (5) was chromato graphed on a Dowex I column (6). Most of the radioactivities were detected in the glucose-6-phosphate fraction. The effect of various substrates on phos phorylation is shown in Table II. The P/ N03- ratio with glutamate was 0.65 which was a fairly high value. Although the P/ NO3- ratio with NADH was not very high, considerable phosphorylation was observed with NADH as the electron donor. When S8 was further fractionated into fractions P8-144 and S144, neither fraction alone had appreciable nitrate reduction or phospho rylation activity, as shown in Table III. However, on combination of the two frac tions, the two activities increased greatly. Fraction P8-344 of E. coli differs from that of P. aeruginosa (2) in that the latter had high nitrate reductase [EC ] activity though not phosphorylation activity, whereas the present particulate fraction had neither. Further purification of S144 is now in pro gress using a Sephadex column, and the pro perties of the S144 fraction will be described elsewhere. As shown in Table IV, inhibition of the phosphorylation coupled with nitrate reduc tion was detected in the presence of 2,4-di nitrophenol (DNP) at a concentration of 10-5M. This inhibition by DNP increased with increase in the concentration of DNP. Phos phorylation was also inhibited in the presence

3 Nitrate Respiration. II 133 Effect of Various Substrates on Phosphorylation with Fraction S8 II For reaction conditions, see the legend for Table I. Fraction SB was dialysed against 0.1 M KCl con taining M Tris buffer (ph 7.4) for 2 hours and contained 9.3 mg. of protein per ml. III Reconstruction of Phosphorylation and Nitrate Reduction Activities by Combination of Particulate and Soluble Fractions For reaction conditions, see the legend for Table I. Total volume of the reaction mixture was made up to 1.1 ml. with distilled water. Fractions Se, S144 and P8-144 contained 6.8 mg., 3.0 mg. and 2.6 mg. of protein per ml., respectively. of 10-3 M KCN. Potassium cyanide inhibited not only phosphorylation but also nitrate re duction at a concentration of 10-2 M. Carbon monoxide inhibited neither phosphorylation nor nitrate reduction. As shown in Table V, phosphorylation coupled with nitrate reduc tion was inhibited completely by amytal at a concentration of 10-3 M. Antimycin A also inhibited phosphorylation at a concentration of 10-3 M. However, it is significant that neither amytal nor antimycin A inhibited nitrate reduction. DISCUSSION In the present investigation, oxidative phosphorylation coupled with nitrate respira tion was shown to occur in cell-free extracts of E. coli, as had been shown previously in pseudomonads (2, 3). This suggests that the generation of energy for growth from nitrate respiration is a widespread phenomenon in denitrifying bacteria. E. coli did not use nitrite as a terminal electron acceptor unlike P. aeruginosa in which electrons are transferred to nitrite via a cy tochrome system (7, 8). As the P/N03- ratios were 0.65 (Table II) and 1.1 (Table V) with glutamate and citrate, respectively, as electron donors, it is concluded that 1 mole of ATP is formed during the reduction of 1 mole of nitrate to nitrite in cell-free extracts of E. coll. The effects of KCN and DNP on phospho rylation coupled with nitrate reduction in cell-free extracts of E. coli are very similar to their effects on phosphorylation coupled with aerobic oxidation of these substrates (9). However, the effects of antimycin A and amytal on oxidative phosphorylation coupled with nitrate reduction are very different from

4 134 A. OTA, T. YAMANAKA and K. OKUNUKI Effect of DNP and XCN on Phosphorylation with Fraction S8 For reaction conditions, see the.legend for Table I. Fraction S8 contained 3.4 mg. of protein per ml. Effect of Amytal and Antimycin A on Phosphorylation with Fraction S8 V For reaction conditions, see the legend for Table t. Fraction S8 contained 4.0 mg. of protein per ml. their effects on oxidative phosphorylation coupled with aerobic oxidation of these sub strates in that these inhibitors are only uncouplers in nitrate respiration whereas they inhibit electron transfer during the aerobic oxidation of these substrates. These facts in dicate that these inhibitors do not inhibit electron transfer between the substrates and the nitrate reductase in cell-free extracts, while amytal is known to inhibit a purified nitrate reductase partially (10). As it is known that electrons are transferred from substrates to nitrate reductase via cytochrome b1 (which corresponds to mammalian cytochrome b) (10), the above facts are compatible with the idea that antimycin A acts on the sites between cytochromes b and ct in mammalian mito chondria (11), but conflict with the idea that amytal acts on sites at a normal redox potential value below that of flavoprotein (11). It is very interesting that these inhibitors act as uncouplers even when they do not inhibit electron transfer. It was shown that in cell-free extracts of E. coli as in those of P. aeruginosa (2) both particulate and supernatant fractions separat ed by centrifugation at 144,000?g were neces sary for phosphorylation. This is similar to the case in aerobic oxidative phosphoryla tion where it is known that the combination of a particulate and a supernatant fraction is necessary for phosphorylation in cell-free extracts of various bacteria (9). SUMMARY Oxidative phosphorylation coupled with anaerobic nitrate reduction was found to occur in cell-free extracts of Escherichia coli. The P/NO3- ratios during the reduction of nitrate to nitrite were 0.65 and 1.1 with glutamate and citrate, respectively, as electron donors. Phosphorylation coupled with nitrate

5 Nitrate Respiration. II 135 reduction was inhibited in the presence of KCN and 2,4-dinitrophenol but not in the presence of CO. Antimycin A and amytal acted as uncouplers of phosphorylation cou pled with nitrate reduction. The authors wish to express their thanks to the Public Health Research Institute of Kobe City, Kobe for its generosity in providing the strain of E. coli used here. REFERENCES (1) Sato, R., Sci. Rev., 2, 122 (1950) (2) Yamanaka, T., Ota, A., and Okunuki, K., J. Biochem., 51, 253 (1962) (3) Ohnishi, T., J. Biochem., 53, 71 (1963) (4) Hersey, D.F., and Ajl, S.J., J. Gen. Physiol., 34, 295(1950) (5) Hagihara, B., and Lardy, H.A., J. Biol. Chem., 235, 889 (1960) (6) Khym, J.X., and Cohn, W.E., J. Am. Chem. Soc., 75, 1153 (1953) (7) Yamanaka, T., Ota, A., and Okunuki, K., Biochim. et Biophys. Acta, 53, 294 (1961) (8) Yamanaka, T., and Okunuki, K., Biochim. et Biophys. Acta, 67, 379 (1963) (9) Brodie, A. F., J. Biol. Chem., 234, 398 (1959) (10) Itagaki, E., Fujita, T., and Sato, R., J. Biochem., 52, 131 (1962) (11) Chance, B., in " Enzymes: Units of Biological Structure and Function", ed. by O.H. Gaebler, Academic Press Inc., New York, p. 447 (1956)