Phosphorylation associated with nitrate and nitrite reduction in Micrococcus denitrificans and Pseudomonas denitrificans

49°

BIOCHIMICA ET BIOPHYSICA ACTA

BEA 65340 PHOSPHORYLATION ASSOCIATED WITH NITRATE AND NITRITE REDUCTION IN MICROCOCCUS DENITRIFICANS AND PSEUDOMONAS DENITRIFICANS M, S. NAIK' AND D. J. D. NICHOLAS Department of Agrioultural Biochemistry, Waite Agricuitural Research Instit-ute, University of Adelaide, (SOtttlt A ustralia} (Received July 5th, 1965)

SUMMARY In cell-free extracts of Micrococous denitrificans, phosphorylation associated with the reduction of nitrate was observed with NADH:/" succinate, malate, pyruvate or glutamate as hydrogen donors. Nitrite reduction was also shown to be coupled to phosphorylation, but nitric and nitrous oxides and hydroxylamine were ineffective. Specific activities of enzymes concerned with electron transfer to nitrate (NADH 2 : nitrate oxidoreductase, Ee r.6.6.r), to nitrite, to cytochrome (NADH:/,:cytochrome c oxidoreductase, EC 1.6.2.r) and to oxygen (cytochrome c:O:/, oxidoreductase, EC 1.9.3.r), which were coupled to phosphorylation were maximal in the particulate fraction collected between 70000 to 144000 X g. Phosphorylation linked to the reduction of nitrate or nitrite in these particles was inhibited by 2,{-dinitrophenol, arsenate, cyanide and amytal but carbon monoxide was without effect. In cell extracts of Pseudomonas denitrificans, however, phosphorylation did not occur with nitrite as the terminal acceptor.

INTRODUCTION A phosphorylation coupled to nitrate respiration in facultative anaerobes has been reported by YAMANAKA, OTA AND OKUNUKI 1 , 2 , WHATLEy 3 , OHNISHI 4 and HEMPFLING, STEINBERG AND ESTABROOK 5 • Micrococcus denitrijicans grows anaerobically provided nitrate is supplied in the culture medium and a mechanism for electron transfer from H 2 or NADH:/, via FAD and cytochromes to either oxygen OJ;, nitrate has been proposed by FEWSON AND NICHOLAS 6 . In this paper the effects of various hydrogen donors and acceptors on anaerobic phosphorylation in cell-particles of this bacterium are described. It is shown that phosphorylation is associated with the * Colombo Plan postdoctoral fellow on leave from the Indian Agricultural Research Institute, New Delhi, India.

Biochim, Biopltys. Acta, lI3 (1966) 490-497

PHOSPHORYLATION IN M. denitrificans AND Ps. denitrificans

491

reduction of nitrite as well as that of nitrate but nitric oxide, nitrous oxide or hydroxylamine were ineffective. EXPERIMENTAL Materials and methods Cytochrome c, ADP, NADH 2 , NADP, hexokinase (EC 2.7.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were obtained from Sigma Chemical Company, Missouri, U.S.A. Reduced cytochrome c was prepared from mammalian cytochrome c by reduction with hydrogen gas in the presence of palladised asbestos. Carrier-free radioactive orthophosphate was obtained from the Atomic Energy Research Establishment, Amersham, Great Britain. Preparation of cell-free extracts and particles Cells of M. denitrificans (N.C.I.B. 8944) grown in modified Grohman's medium under nitrogen gas at 30° for 20 h, were harvested in a Servall continuous flow centrifuge at 4° and washed free of nitrite with 0.85% NaCl. The washed cells suspended in 0.05 M Tris-HCl buffer (pH 7-4) (I cell t r buffer) were disrupted by repeated freezing and thawing (six times) in a dry ice acetone mixture and centrifuged at 20 000 X g for 30 min at 0°. The supernatant fraction was used as the cell-free extract and the following particulate preparations were separated from it: 20 000-35 000 X g for IS min (A), 35 000-70000 X g for IS min (E) and 70 000-144 000 X g for 2 h (C). Cells of Pseudomonas denitrificans (A,T.C.C. 13867) were similarly grown and the following cell-free preparations were made: (I) crude extract: 20000 X g for 30 min, supernatant fraction, (2) particle-free supernatant fraction: 144 000 X g for 2 hand (3) particulate fraction: 144 000 X g for 2 h. incorporation and determination of ATP The incorporation of 32p was followed under anaerobic conditions in Thunberg tubes. The reaction mixture in a final volume of 1.5 ml contained in ,umoles: 25 TrisHCI buffer (pH 7-4), I MgC1 2, 5 NADH 2 or other Hz donors, I ADP, 20 NaF, 10 Pi (18 000 counts/min 32p), I KN0 3 , NaN0 2 or NH 20H-HCl and 0.1 ml enzyme (5 mg protein). After IS min incubation at 30° 0.5 ml 20% HCl0 4 was addecl. The 32p incorporated into nucleotides was assayed by adsorption onto charcoal discs by the method of MORTON, RAISON AND SMEATON 7• order to study the effect of nitrous and nitric oxides on phosphorylation, the Thunberg tubes were evacuated and filled up with these gasses. ATP was also assayed by the glucose-hexokinase-glucose-6-phosphate dehydrogenase-NADP method, hereafter called the NADPH 2 method. The reaction mixture was as indicated above except that hexokinase (14 Kunitz-McDonald units) and 20 ,urn ales glucose were added. After IS min incubation at 30°, 0.5 ml 0.2 N HCI was added and the tubes immersed in a water bath at 100° for I min, to oxidise the residual NADH 2 • The supernatant fraction obtained after centrifuging at 2500 X g for 3 min, was adjusted to pH 7-4 with 0,1 M Tris (pH r r). The glucose-fi-phosphate formed in the reaction was assayed in a suitable aliquot in the following 3 ml reaction mixture: 5o,umoles Tris-HCI buffer (pH 7-4), I20 units glncose-6-phosphate dehydro-

32p

In

B-iochim. Biopliys. Acta, II3 (r9 66) 490--497

M.

S. NAIK,

D.

J.

D.

NICHOLAS

genase, 4 ,umoles MgCl2 and 0.4 ,umoles NADP. The increase in absorbancy at 340 mp was determined in a Unicam SP700 recording spectrophotometer. The [32PJATP-exchange reaction was measured in the following reaction mixture in a final volume of 1.5 ml in ,umoles: roo glycylglycine buffer (pH 8.0), 10 MgCI 2, 10 Pi (pH 8.0) (containing 32p 60000 counts/min), 4 ATP and 0.1 ml of bacterial extract. After IS min incubation at 30° the [32PJATP formed was counted after adsorption onto charcoal discs. Enzyme assays Nitrate reductase (NADH 2:nitrate oxidoreductase, EC 1.6.6.1) was assayed by the method of Fzwsox AND NICHOLAS8. The reaction mixture in I ml contained I pmole KN0 3, 70 pmoles phosphate buffer (pH 7-4), 0.1 ml enzyme and 0.5 ,umole NADH 2. After 10 min incubation at 30°,0.1 ml M zinc acetate and 1.9 ml95 % ethanol were added, the mixture thoroughly shaken and then centrifuged at 2500 X g for 5 min. Nitrite was determined in an aliquot of the supernatant solution by the Greiss-Ilosvay colorimetric method. Nitrite reductase activity was assayed in the following reaction mixture in Thunberg tubes in a final volume of I ml: 0.5 ,umole NaN0 2, 70 pmoles phosphate buffer (pH 7.4),0.1 ml enzyme, 0.1 pmole FAD and 0.25 ftmole NADH 2. After 30 min incubation at 30° residual nitrite was determined by the method described above. NADH 2-cytochrome c reductase (NADH 2:cytochromec oxidoreductase, EC 1.6.2.1) was assayed under anaerobic conditions in I-cm cuvettes fitted with Thunberg attachments. The reduced cytochrome c formed in the reaction at 30° for IS min was determined from the increase in absorbancy at 550 m,u in a Unicam SP700 recording spectrophotometer. The reaction mixture in 3 ml contained: 125 pmoles Tris-HCI buffer (pH 7-4), 0.5 pmole NADH 2, 0.1 pmole cytochrome c in the tube and 0.1 ml of the enzyme in the side arm. The reaction was started by tipping in the contents of the side arm. Cytochrome c oxidase (cytochrome C:02 oxidoreductase, EC 1.9.3.1) was assayed in open cuvettes in the recording spectrophotometer by following the oxidation of reduced cytochrome c. The reaction mixture in a final volume of 3 ml contained 130 zzmoles Tris-HCI buffer (pH 7-4), 0.1 pmole reduced cytochrome c and 0.1 ml enzyme. The decrease in absorbance was measured at 550 mf-l for 15 min at 30°. Oxygen uptake was measured with a Beckman oxygen electrode. Protein was determined by the Folin method". RESULTS Even in the absence of any terminal acceptor a considerable amount of 32p was incorporated with the various hydrogen donors (Table I). This amounted to between 30 and 70% of the total 32p incorporated when nitrate was present. A particle-free supernatant fraction obtained after centrifuging the cell-free extract at 144000 X g for z h contained an active adenylate kinase. TheATPformed during this reaction was assayed by the NADPH 2 method and the results are shown in Table II. The production ofNADPH 2was proportional to the ADP added. The supernatant and the particulate fractions also contained ATP-32P exchange activity at 75 and 31 m,Lfmoles of [32PJATP/mg protein/IS min, respectively. Thus the 32p incorporation without a Biochim: Biopbys. Acta, lI3 (I966) 490-497

M. denitriflcans

PHOSPHORYLATION IN

AND

PS. denitrificans

493

TABLE I EFFECT OF VARIOUS HYDROGEN DONORS ON INCORPORATION OF 32p IN CELL FREE EXTRACTS OF

M. denitrificans Reaction at 30° for IS min. Complete reaction mixture in a total volume of I.5 rnl, in pmoles: 25 Tris-HCI buffer (pH 7.4), I MgCl a, 5 NADH. or other H a donors, I ADP, 20 NaF, 10 Pi (18 000 counts/min 3'P). I I(NO a or NaNO a and 0.1 rnl enzyme (cell-free supernatant fraction left after centrifuging at 20 000 X g for 30 min). [32PJATP formed was assayed as described in the METHODS.

mflmo!es of P; incorporated ~"

Hydrogen donors

_ _ _'-u_

Terminal acceptor

NADH 2 Succinate Malate Pyruvate Glutamate

None

Nitrate'

Nitrite'

Oxygen'

144 14 8

34 0 122 26 5 25 0 105

3 16 178 JIg 116 12 4

201 17 8 16 5 162

2°4

18 7 196

211

._.~--~--"--

, These values corrected for those obtained without a terminal acceptor.

/_----~--------o /""

0.4

0

0/

/

/

/

/

/

I

/

/

I

i

0.3

I

/0 / /

x~x.~--x-

/

U

QJ

;;:

/l

L 0.2

QJ

/

// /X

+"'

2

a:

/

2

~ 0.1

I

/0

,t x.:

/1

za,

/~/

lx 0'

o

2 urnoles NOj

3

or

4

NO~

Fig. 1. Effect of graded amounts of nitrate or nitrite on phosphorylation in M. deniirifican«, The reaction mixture as described in Table 1. 0- - - - - 0, nitrate; X - x , nitrite.

Biochim, BioPhys. Acta, 113 (1966) 490-497

M. S. NAIK, D.

494

J.

D. NICHOLAS

TABLE 11 ADENYLATE KINASE IN THE PARTICLE-FREE SUPERNATANT FRACTION

Reaction at 30° for 15 min. The reaction mixture in a total volume of 3 rnl in !lmoles: 50 Tris -H'Cl buffer (pH 7.4),4 MgCI2 , ADP as indicated, 20 glucose, hexokinase (14 Kunl tz -Mc Donald units), glucose-6-phosphate dehydrogenase (120 units), 0.4 NADP, and 0.1 ml of enzyme (supernatant fraction left after centrifuging at 144°00 X g for 2 h). The NADPH. formed was determined by the increase in absorbancy at 340 m,u in the SP700 recording spectrophotometer.

ADP added NADPH 2!ormed (mflmoles) (flmoles to the reaction mixture} o 0.25

o 43

I.O

rr6

2·5

188

terminal acceptor resulted in part from adenylate kinase activity and a subsequent ATP_32P exchange reaction. When these values were deducted from those obtained in the presence of a terminal acceptor, the phosphorylation values listed in Table I were obtained with various donors. The best one was NADH 2 and with it phosphorylation was proportional to the amounts of nitrate or nitrite added as shown in Fig. 1. At equivalent concentrations, nitrate was more effective than nitrite. Phosphorylation did not occur when nitric oxide, nitrous oxide or hydroxylamine was used in place of nitrate or nitrite. Oxidative Phosphorylation in the particulate fractions Maximum 32p incorporation coupled to nitrate or nitrite reduction was obtained with the particulate fraction C collected between 70 000 and 144 000 X g when NADH 2 was the donor (Table III). Malate or succinate did not substitute for NADH 2. The production of ATP was also confirmed by the coupled NADPH 2 method (Table IV). TABLE III PHOSPHORYLATION ASSOCIATED WITH NITRATE AND NITRITE REDUCTION IN PARTICULATE FRACTIONS OF M. deniirifioans

Enzyme assays as in Table I. Particulate fractions were made as described in umoles Pi esterified] mg protein

Particulate fractions Terminal acceptor collected at uarious g values Nitrate Nitrite

- - - - - - - - - - - --------A (20000- 35000) B (35 000- 70 000) C (70000-144000)

0.15

nil

0·33

0.3 1

I.gI

1.54

Biochim. Biopbys. Acta, II3 (Ig66) 490-497

METHODS.

PHOSPHORYLATION IN

M. denitrijicans

AND

PS. denitrifican«

495

TABLE IV ATP FORMATION IN TlIE PARTICULATE FRACTION (C) TESTED

BY

THE NADPH 2 METHOD

Complete reaction mixture in a total volume of 1.5 ml in ,umoles: 25 Tris-HCI buffer (pH 7.4) I MgCI 2, 5 NADH z' ADP as indicated, 20 NaF, 10 PI, T KN0 2, 20 glucose, hexokinase (14 units). Reaction in Thunberg tubes at 300 for 15 min. Glncose-o-phosphate formed was assayed by the NADPH 2 method as described in METHODS.

Reaction mixture

NADPH 2

formed {mumoles )

Complete (I flmole ADP) Complete (2.5 flmoles ADP) 2·5 flmoles ADP (omit NADH z and KNO.)

29

TABLE V SPECIFIC ACTIVITIES OF NITRATE, NITRITE, NADHz-CYTOCHROME ClIROME OXIDASE IN PARTICULATE FRACTIONS OF M. denitrificans

C

REDUCTASES AND OF CYTO-

Enzyme assays as in METHODS. Nitrate reductase: mjzmoles nitrite producedjrng protein/min. Nitrite reductase: mzemoles nitrite rcducedjmg protein/min. N ADH 2-cytochrome c reductase: mrzmoles cytochrome c reducedjrng protein/min. Cytochrome c oxidase: mrrmoles cytochrome c oxidisedjrng protein/min.

Particulate fractions collected at various g values

A (20 000- 35000 X g) B (35 000- 70 000 X g) C (7 0 000-144 000 X g)

NADH 2 Nitrate Cytochrome c Nitrite reductase reductase cytochrome c oxidase reductase

80

0.3

0.2

93

16

0.8

1.1

30 0

89

1.6

8.0

TABLE VI EFFECT OF INHI13ITORS ON PHOSPHORYLATION ASSOCIATED WITH NITRATE AND NITRITE REDUCTION IN M. denitrijicans

% Inhibition. Terminal acceptor

Inhibitors (M) Din-itrophenol

A rsenate

A mytal

Potas-

t or?

IO-'

siurn

Carbon monoxide

cyanide

IO-4

10- 3

Nitrate Nitrite

82 97

100 100

37

69

58 33

Nil Nil

Biochim, Biophys. Acta, 113 (19 66) 490-497

M. S. NAIK , D.

J. n.

NICHOLAS

Specific activities of nitrate, nitrite and NADH 2-cytochrome c reductases as well as that of cytochrome oxidase were higher in pa rticulate fraction C than in those A and B (Table V). In fraction C with NA DH 2 as the do nor, phosphorylation associated with nitrate or nitrite reduction was markedly inhibited by 2,4-dinitrophenol and completely so by arsenate (Table VI). Partial inhibition of phosphorylation was also observed with amytal and cyanide. Carbon monoxide did not ha ve any effect on phosphorylation or on nitrate and nitrite reductases, but it inhibited oxygen uptake (as measured by oxygen electrode) of t he particles by 55 %. This inhibition was partially relieved by exposing to a tungsten light for IO min . Phosphorylation in P . denitrificams Phosphorylation associated with ni t rat e and nitrite reduction was also studied in another facultativ e anaerobe, P. denitrificans (Table VII). In t h e particulat e frac TABLE V ll PHOSPHORYLATION ASSOCIATED WITH NITRATE AND NITRITE REDUCTION AND THE ACTIVITIES OF NITRATE, NITRITE , NADH 2- CY T OC HR OME C REDUCTASES AND CYTOCHROME OX IDASE IN CELL FRACTIONS OF P. denitrificans

Enzyme assays as in METHODS. Phosphorylation: (NADPI-!2 method) flmoles of NADPH 2 formed/ mg protein. Nitrate reductase : rnumoles nitrite produced/mg protein/min . Nitrite reductase : rmsmoles nitrite reducedjrng protein/min. NADH.-cytochrome c reductase : mzzmoles of cytochrome c reducedjmg protein/min . Cytochrome c oxidase : mumoles of red uced cytochrome c oxidisedjmg protein/min.

Phosphorylation with two terminal acceptors

Supernatant (144 000 x g for 2 h) Particulate fraction (144000 X g for 2 h)

NADH., Nitrate Nitrite Gvtochrome c reductase reductase cytochrome c oxidase reductase

Nitrate

Nitrite

Nil

Nil

0 .6

3·7

14. 2

3:1

Nil

17·7

0.1

15·9

37 ·7

I .I

5

t ion, nitra t e red uction was found to be coupled t o phosphorylation, in agreement with the earlier findings of OHNISHI4., but no phosphorylat ion could be det ected when nitrite was the acceptor. Nitrite reduct ase was not found in the pellet but was present in the super nat ant fraction . Nitrate redu ctase and cytochrome oxidase were found mainly in the particles, whereas NADH 2-cytoc11rome c reductase was present in the supernatant fraction as well. DISCUSSION

In M, denitrificans, oxygen or nitrate serve as alternative terminal acceptors in respiration and phosphorylation occurs with bo th. It is shown here that nitrite reduction is also coupled to phosphorylation although to a lesser extent than that for nitrate. Although nitric oxide is reduced to nitrogen gas by t his bacterium, it was ineffective for phosphorylation as was nitrous oxide or hydroxylam ine . This suggests that Biochim , Biopliys. Acta, 113 (1966) 490-497

PHOSPHORYLATION IN

M. denitrificans

AND

PS. denitrificans

497

esterification of phosphate occurs only when inorganic nitrogen compounds containing at least 2 atoms of oxygen are used as terminal acceptors. Phosphorylation obtained with nitrite reduction is of interest since YAMANAKAIO has shown that cytochrome oxidase and nitrite reductase in P. aeruginosa are identical. AZOULAyll has shown, however, that this organism contains two types of cytochrome oxidases; cytochrome oxidase/nitrite reductase (Pseudomonas cytochrome c 551 :nitrite, O2 oxidoreductase) which does not act on mammalian cytochrome c and the other type (Pseudomonas cytochrome c 551: O2 oxidoreductase, EC 1.9.3. I), which can oxidise reduced mammalian cytochrome c but does not reduce nitrite. In M. denitrificans, VERNON AND WHITE12 have shown that the cytochrome oxidase in the particulate fraction oxidised reduced mammalian cytochrome c as well as the bacterial cytochrome. We have found that although carbon monoxide inhibited oxygen uptake, it had little effect on nitrite reductase or on the phosphorylation associated with it. This would suggest that nitrite reductase and cytochrome oxidase may not be identical in this organism. The nitrite reducing system of M. denitrijicans appears to be different from that of P. denitrificans (ref. 13). In the latter it is a soluble enzyme, which explains the absence of phosphorylation with nitrite as the terminal acceptor. ASAN0 14 also found that nitrite reductase in a halotolerant Micrococcus (strain 203) shows important differences from the enzyme in P. denitrijicans. Our results thus provide experimental evidence for the suggestion made by Y AMANAKA10 that in the latter organism nitrite reduction may not be coupled to phosphorylation.

ACKNOWLEDGEMENTS

One of us (M.S.N.) thanks the Colombo Plan Authorities in Australia for a postdoctoral award and the Indian Agricultural Research Institute for leave of absence which made it possible to collaborate in this work. REFERENCES I T. YAMANAKA, A. OTA AND K. OKUNUKI. j. Biochem. Tokyo, 51 (I962) 253. 2 T. YAMANAI(A, A. OTA AND K. OKUNUKI, Abstr. 6th Intern, Congr. Bioohem.; New Y01'k, I964, Part X, p. 750. 3 F. R. VVHATLEY, Plant Physioi. Proc, Ann. Meetings, 37 (1962) viii. 4 T. OHNISHI, j. Biochem, Tokyo, 53 (1963) 71. 5 "V. P. HEMPFLING, S. STEINBERG AND R VV. ESTABROOK, A bslr. 6th Intern: Congr . Biochem.,

New Yoyk, I964, Part X, p. 779. 6 C. A. FEWSON AND D. J. D. NICHOLAS, B·iochim. Biophys. Acta, 48 (Ig6I) 208. 7 R. K MORTON, J. 1<. RAISON AND J. R. SMEATON, Biochem. j., 91 (I9 64) 54 I. 8 C. A. FEWSON AND D. J. D. NICHOLAS, Biochim. Biophys. Acta, 49 (I9 6 I) 335· 9 O. I-I. LOWRY, N. J. ROSEBROUGH, A. L FARR AND R. J. RANDALL,.f. Bioi. Chern., 193 (I95 I) 26 5 . IO T. YAMANAKA, Nature, 204 (I964) 253. II E. AZOULAY, Biochim, Biophys, Acta, 92 (I964) 458. 12 L. P. VERNON AND F. G. WHITE, Biochim, Biopbys, Acta. 25 (I957) 3 2 I. 13 H. IWASAKI. S. SHIDARA, H. SUZUKI AND T. MORI . .f. Biochem. Tokyo, 53 (I9 63) 299· 14 A. ASANO, J. Biochem. Tokyo, 46 (I959) 7 8 r.

Biochim, Biopbys, Acta, II3 (19 6 6) 49°-497