The mechanism of nitrite oxidation by Nitrobacter winogradskyi

BIOCHIMICA ET BIOPHYSICA ACTA BBA

6S3II

THE MECHANISM OF NITRITE OXIDATION BY NITROBACTER WINO-

GRADSKYI

A. VAN GOOL ANlJ H. LAUDELOUT Institut Agronomique, Uiiiuersite de Louuain, Louuain (Belgium) (Received May 28th, I965)

SUMMAHV I. With exception of certain narcotics, the effect of classical inhibitors on the nitrite respiration of Nitrobacter ioinogradskyi was in agreement with the usual inhibition pattern characteristic of a cytochrome respiratory chain. Strong inhibition by quinacrine and its reversal by flavin-adenine dinucleotide shows that a flavin is involved in the nitrite oxidase system. 2. Analysis of the nitrite oxidase system by differential, direct and lowtemperature spectra pointed to the presence of cytochromes of the a and c type, and a flavin component. Cytochrome b was not detected. The cytochrome of the c type was partially soluble, with spectrophotometric properties equivalent to common bacterial cytochrome c. Several cytochromes of type a appeared to be present; CO difference spectra revealed cytochrome a and a 1. 3. As compared with the normal respiratory activity of the nitrite oxidase system, a rather high cytochrome concentration was found. This was considered in the light of the thermodynamically unfavorable first step of the nitrite respiration.

INTRODUCTION The mechanism by which Nitrobacter oxidises nitrite to nitrate with oxygen as the terminal electron acceptor is still far from satisfactorily understood although considerable progress has been made in the last few years. Recognition that cytochromes of the c and a types are implicated in the oxidation results from the work of LEES AND SIMPSONI, BUTT AND LEEs 2, ZAVARZIN 3 and SILVER 4 . The results that these authors obtained on cell suspensions of Niirobacter winogradskyi were confirmed and extended by ALEEM AND NASON 5 working on intact cells and cell-free extracts of N. agilis. Some of the main problems which remain to be solved include those related to the thermodynamics of a cytochrome-linked oxidation of nitrite, as recently discussed by LEES6 , and those pertaining to the nature and succession of the steps by which electrons are carried from nitrite to oxygen. Biocbim. Biophys, Acta, II3 (I966) 4I-50

A. VAN GOOL, H. LAUDELOUT METHODS

The cells were grown, harvested and washed according to the techniques outlined by BOON AND LAULJELOl'T 7. Cell-free extracts were obtained by ultrasonic treatment of 35 ml of concentrated washed cell suspensions (about 300 mg dry wt.) for a period of 60 min in a MSE (io \V, 20 kcycles/sec ultrasonic disintegrator. The cell suspension was placed in an ice-jacketed tube under a N 2 atmosphere during the ultrasonic treatment. Centrifuging at 6000 ;< g during 10 min at 0° in a Spinco Model L centrifuge was sufficient to remove all intact cells, since no growth could be obtained from the supernatant. All respiration measurements for determining the effect of inhibitors were made with the usual \Varburg technique with 0.2 ml of the cell suspension or cell-free extract and r.8 ml phosphate buffer (IS m11 Na 2HP04- 2 m'M KH 2P04 , pH 7.6) in the flask to which 0.5 ml of the inhibitor solution was added. NaN0 2 was added from the sidearm as 0.5 ml of a 96 rrrM solution, to give 16 mM final concentration which is optimal for nitrite oxidation". Difference spectra of cell suspensions or cell-free extracts were observed with the Beckman DK-I double-beam spectrophotometer. The cuvettes were maintained at 30° and contained 3 ml of cell suspension or cell extract in phosphate buffer (pH 7.6). The contents of one cuvette were reduced with either a few crystals of Na 2S204 , or 10 ftl of a N0 2 - solution (final concentration 20 mM N0 2 -) before registering the difference spectra. Direct spectra were obtained with either the phosphate buffer solution, or, if the cell-free extract was too turbid, 'with a piece of filter paper in the reference path. TABLE I INHIBITION OF NITRITE OXIDATION BY NITROBACTER

NO s- concentration 16 mM. Duration of thermal equilibration period and consequently of the incubation of the substrate-free system with the inhibitor: 15-30 minutes. Temp. 30°. pH 7.6. No KOH in center well. The 50% inhibition concentration for HNO s is calculated from the results of BOON AND L.<\UDELOUT' on the pH vs. inhibition curves. Ethylurethane and amytal gave no inhibition at 1.0 rn M concentration. Antimycin A at a concentration of 20ltg/ml of cell-free extract gave 60% inhibition. Inhibitor

50% inhibition concentration

tc, (mM)

(mN!)

Cells

KCN NuNs

0.04 0.01 0.02

HNO s KCNO 2.j 1.0 NHsOH KCl0 3 to Quinacrine· ncr p-Hydroxymercuribenzoate Iodoacetamide 10 2.4-Dinitrophenol 0.5 a,u'-Dipyridyl 1.0 o-Phenanthroline 1.0 BAL

Biochim, Biophys. Acta, II3 (1966) 41-50

Cell-free extract o.ooq

o.ooy 0.01 7 3 0.04 0.04 0.2

3°

o.q

10 0')

0.003

0.0 19 0.4 8

ME CHANISM OF NITRITE OXID ATION BY NITROBACT ER

43

RESULTS

I nhi bitor studies Some of the evidence which m ay be found in the literature relati ve t o the effect of imp ortant met abolic inhibit ors on the rate of nit rite oxidat ion by Nitrobacter is conflicting , as discussed further in this paper. Thi s has pr ompt ed us t o re-examine quant it atively the effect of the fairly comprehensive list of inhibitors shown in Table 1. The inhibition refers t o the rat e of oxygen uptake as measured in the Warburg apparatus on cell suspensions or cell-free extracts, the Q02 of which was about 500,a1 O 2 per h per mg dry matt er or about 6000 ItI O2 per h per mg N. The QE{2of cell-free extracts was about 400 ttl per h per mg N . Th e inhibitor concentrat ion giv ing 50 %) inhibition has been calculated by interpolat ing on the inhibition percentage vs. inhibitor-concentration curve established for at least four concentrations corresponding to partial inhibition. For some inhibitors, the relative inhibition was studied at two substrate concentrations (r6 111M and 6 111M) . These results were plotted according to the method of DIXONS which perm its cha ruc t eriza tion of th o inhibition and calculation of the Michaelis constant ](1 of the inhibitors. Table I records t he observat ions made on 13 inhibitors expressed as t he 50 % inhibition concentration toget her with th e Micha elis constant 1(\ for cert ain of the inhibitors. F u rthermore. special at tent ion was paid to quinac rine with respect to th e reversal of its action by FAD . Th e results of this reversal study are presented in Tabl e II. TABLEII

F Ail

IHWE IUii\ L O il ~ ll INi\( ~IUN" I N ll l lllT [()N 01' NITIW Il i\CT1m N I'l'R ITl' TtllSl'tRi\l'IO N

Con ditio ns: 0 . ) m l of n trw 111M nltri tc so lutio n was ad cl"d from t he side -a rm t o 0.3 ml cell- fr ee extract. 0. 3 ml of a solu t lon of FA 1) a n djor qu inac r ine to give des ired fin al conc ent rations and 2.1 111 1 of Tris buller (pI [ 7.CI). Temp. 3o G.

Quinacrine - Il Cl FA D conclI. concn , (j tivl) (I u'll)

0

0

60 60

0

60

R ate of 0 2 upta k«

It// II per 1111 cell-free extract

R elative rates

32 2 12 9 39 0

100 40 122

Spectrophotometry oj the ni tri te oxidase system Most of the spectrophotometric information presented below was obtained in t he form of differ ence sp ectra : two ident ical cell suspensions or cell-free extracts ar e pl aced in the light paths of the double-beam spectroph ot omete r. Adding an appr opriate reducing agent to one of th em changes its transmittance. This change is recorded in a range of wavel ength from 400 to 650 mp, and expressed as the rati o of t he transmittance of th e reduced suspension to t hat of the oxidized suspension at the same wa velength. Fig. r sh ows such a difference spect ru m for an intact cell suspension (A) and a cell-free extract (B) which had been reduced by N0 2- or S2042-. A clear er picture B ioclii m, Biopbys . Acta, II 3 (1966) 41-5°

44

A. VAN GOa L. H. LAUDELOUT 8

0 .6 0 .6

0.8

1.0

'" u

1.0

~

~

A

E

c o'" L

E

e o'"

.... 0 .90

0 .93

0.95

0 ,9 5

,00

0,9 7

40 0

440

480 520 560 Wave l engt.h (rnu)

600

'"

~

,=

670

F ig. I . Difference spectra of Ni trobacter cell suspension a nd cell-free extract. Th e bas e line was recorded w ith the oxid ized cell su spension (A) or cell-free ext ra ct (B) in both light paths; the red uced minus oxidized spectra ob t ained a ft er NO.- an d 8 2 1' " reduction, are represented by dotted a nd fu ll lines r espect iv ely .

°

resulted from the cell-free extract since the three ba nds in t he visib le region and the two in the Soret region were more pronounced than was t he case for spect ra of cell suspensions. Averaging t he obser vat ions of a fairl y large number (20) of difference spectra has led to th e following cha racterist ic values of the absorption maxima and the isosbestic points. In the visible, maxima were found at 523,554 and 597 mp. wit h a shoulder at 609 m/-l on the latter. A broad zone was found between 450 and 490 mp. with a minimum at 465 111ft in which the transmittance of the reduced system was higher than that of t he oxidized system. In the Soret region. two peaks were observe d at 419 and 439 rnp. which, as noted earlier, wer e fus ed in a single peak at 440 mft in the case of turbid cell suspensions. When cell susp ensions are compared with cell-free extra ct s, slight differences between the average positions of the absorption maxima may be found in the difference spectra. This was most commonly observed for the peak which occurred at 597 m,a for the cell susp ension and was found at 594 mp. in th e case of the cell-free extract. The localizations of the isosbestic points fluctuated mu ch more than those of the absorption maxima. T wo values of isosbestic points seemed, however. fairl y reliable within 1-2 mp., nam ely 580 and 543 mp. in the case of cell suspensions. Th e relative intensities of the peaks in the difference sp ectrum of a dithionite-redu ced cell suspension were 1.0 :1.21 :0.37 :3.8 for the peaks a t 597.554.523 and 439 mft , respectively . Th ese relative intensities in the case of a cell-fr ee extract were quite different at I : 2 .1; 0·5 :4,55 for the bands at 594. 554. 523 and 439 111 f t , respecti vely . Biocnim, Biophys. Acta, 1I3 (196 6) 41-50

MECHANISM OF NITRITE OXIDATION BY NITROBACTER

45

Since the purest cell-free extracts obtained presumably did not reveal the whole complexity of their difference spectrum, a low-temperature spectrum was conducted (Fig. 2). At the lower temperature the highest peak in the visible range was split into

III

u

t:

a

.c

I-

a

l/l

.c

« -0.04 450

500

550

600

Wavelength (mfJ)

Fig. 2. Low-temperature spectrum of a particulate Nitrobacter fraction. A freeze-dried particulate fraction, obtained from a crude cell-free extract after centrifuging at 150 000 X g for 2 h, was examined by Dr. B. CHANCE (Expt. 50 IV),

three distinct peaks with maxima at 604,587 and 579 IDfl and the asymmetry which was observed for the absorption maxima at 594 mit was thus fully resolved. No other modification was observed in the low-temperature spectrum with respect to the room" I,

,

!l 11

,

II,

I

/:

II

I

I I

I

fl

E 0.89

~o

:

I I :

I

I

I

I I

/' I I I I \ ! i' f \ I \ /

I

.=

I

I i

c

"=

I

!\

I"

i;' 0.88

..8

I

'\

0.87

\'

\:

085

I\ : \

II

\)

I

,

I

,

,

I

II

',

I I

,,

0.90

\

, , \

\

\

1.00

I

,

I

I

I

I

I

I

\

r

0.95

\

,

I

\ I

I

\

,--,-

"'----

1.10 .... a

.... 'f

'"

1.15 co

\\j"

\

~

\

I

"

o

c

I f

I

\ O.91 ~--'>.L-\---H-">--1

>,

I

1.05

\

1.05

,

\

I

I

I

\I

I I

I

l"-"~

I

:

1.00

I I

,I....

,

J I

\

\

c

8

I

, ' : :, ,, I

I

1 \,

/

0.86

t

I

0.80

1\

i,

0.85

I

A

""

0.84

1.20 1.15

0.92 500

540

400

420

440

460

400

420

440

Wavelength (mfJ)

Fig. 3. CO difference spectra of Nitrobacter cell suspension and cell-free extract. The base lines were recorded after N0 2- reduction of sample and reference. Full lines correspond to additional absorption in the reference or sample path after bubbling CO, for I min, through the sample cuvette containing cell-free extract (A and B), or cell suspension (C). Dotted lines represent N0 2- reduced minus oxidized difference spectra of the same preparation.

Biochis«. Biophys: Acta, II3 (1966)

41-50

A. VAN GaOL, H. LAUDELOUT

temperature spectra, apart from the well known shift of the absorption maxima toward lower wavelengths. In order to obtain more information about the nature of the respiratory-chain constituents, CO difference spectra were obtained by bubbling a stream of CO gas through one of the two cuvettes containing the nitrite-reduced sample. In the difference spectra presented in Fig. 3, peaks correspond to additional absorption in the sample cuvette, and troughs correspond to additional absorption in the reference path. To facilitate interpretation of the CO difference spectra, reduced vs. oxidized spectra of the same preparation are also presented. The base lines of the former were obtained as the trace recorded with oxidized suspensions in both light paths, whereas the base lines of the CO difference spectra were the traces obtained when the contents of both cuvettes were reduced by N0 2 - before bubbling CO through the sample cuvette. In Fig. 3, A and B refer to cell-free extracts, while C refers to the CO difference spectrum of a cell suspension in the Soret region. The sensitivity of the instrument did not permit close observation of CO difference spectra for intact cell suspensions in the visible region. As shown in Fig. 3A, the most obvious consequence of bubbling CO through the sample cuvette was the immediate increase of the reduction of the pigments as evidenced by the peaks at 523, 554 and 608 mft in the visible, and at 4I9 111ft in the Soret region. Specific effects which resulted directly or indirectly from the CO complexing of the pigments included a trough corresponding to the 439-mfl peak in the reduced vs. oxidized spectrum, a small but reproducible peak at 450 111ft, and a TABLE III ABSORPTION MAXIMA AND RELATIVE BAND INTENSITIES OF THE SOLUBLE NlTROBACTlm FRACTION

Direct spectra were recorded from the supernatant obtained after centrifuging ,1 crude cell-free extract at 150 000 X g for 2 h. The relative intensity is expressed as the ratio of the absorbance at the wavelength indicated to that of the first.

A bsorption maxima a Oxidized Dithionite-reduced

{J

551.5 5 2 2 55 2.5 5 2 3

Relative intensity

y

(5

a

{J

y

4 II

3 2 5.5

La La

0·7

7·4 5·7

4 16

(j

trough corresponding to the 594-m,u peak. When an intact cell suspension was examined, the trough at 440 mp" and a small peak at 426 mft were present, and apparently no increase in the reduction ofthe 419-mp, constituent after CO treatment. The solubility of the components to which the absorption peaks of the difference spectrum are due, was investigated by separating the crude cell-free extract, obtained as described above, into two fractions. Centrifuging at ISO 000 X g for 2 h gave a supernatant devoid of nitrite oxidase activity and a dark red sediment in which the activity of the cell-free extract was concentrated. As the supernatant thus obtained was not turbid, direct spectrophotometric investigation was possible. The results drawn from absorption spectra of the soluble fraction are given in Table III. The sediment obtained from centrifuging the crude cell-free extract, all the Bioohim, Biophys, Acta, II3 (1966) 41-5°

MECHANISM OF NITRITE OXIDATION BY NITROBACTER

47

other hanel, yielded a difference spectrum such as the one in Fig. 4 where all the peaks found in a crude cell-free extract are again observed. With regard to the specificity of the reduction of these respiratory pigments, it should be noted that N0 2- was the only substrate found which led to an appreciable

if

0.6

0.88

0.8

0.92

c

~

E

~

o ~

G c

~

E

\+''>--+-+---,1--_ _''<----71°. 9 6

1.0

§ ,0:

400

440

480 520 560 Wavelength (rnp )

600

640

Fig. 4- Difference spectrum of a particulate Nitrobacter fraction. A crude cell-free extract, was centrifuged at 150 000 X g for 2 h and the pellet resuspended in phosphate buffer after a r-rnin ultrasonic treatment. The base line was recorded from the oxidized suspension in both light patbs, while the difference spectrum was obtained after 4 ' - reduction of the sample.

8.°

reduction. Succinate, lactate, citrate, xanthine did not lead to any appreciable reduction. Bands did appear after addition of formate or NADH 2 and their localization was identical to those of the nitrite reduction bands but their height was only one-third and one-half, respectively, of those observed after nitrite reduction of the respiratory pigments. DISCUSSION

It is superfluous to discuss the effect of the classical respiratory-chain inhibitors, since the cytochromic nature of the respiratory chain of Nitrobacter iswell established. It may be interesting to compare the value of the 50% inhibition concentrations of those inhibitors for intact cells and cell-free extracts. The similarity of the values seems to form circumstantial evidence for the peripheral location in the cell of the enzyme system causing nitrite oxidation. Turning now to a comparison of the values presented here with those of the literature, it is seen that there is a substantial agreement. Results listed for Cl0 3 - in Table I are in accord with the data of LEES AND SIMPSON 1 . These authors found an inhibition of about 50 % after 60 min incubation at 7 roM Cl0 3-, which is comparable to the 50% inhibition found after 30 min incubation at IO mM. The necessity of stating the incubation period for CI0 3 - inhibition stems from fact that the decomposition products of this inhibitor destroy the cytochrome activity according to a first-order rate law which has been studied by LEES AND SIMPSON 1 . Further agreement may be found in the antimycin 50% inhibition which occurs at a concentration very similar to that reported by ALEEiVI AND NASON 9 • The effect of dinitrophenol on the respiration rate is practically identical to that mentioned by BUTT AND LEES2; hence the lack of respiration inhibition by 2,4-dinitrophenol mentioned by ENGEL, KRICK Biochim. Biopllys. Acta, II3 (I966) 4I-50

A. VAN GOaL, H. LAUDELOUT AND FRIEDR1CHSEN 1 0 is difficult to explain. Finally the lack of inhibitory effect exhibited by narcotics is in agreement with the early observations of MEYERHOFl l . The main feature of the data given in Table I is undoubtedly the powerful effect shown by the flavin inhibitor, quinacrine. This is at variance with the findings of other authors on this or other acridine-derivative inhibitors. ZAVARZIN 3 mentions 50% inhibition concentrations of 0.15 mM and 0.12 mM for acrichine and rivanol, respectively. ALEEM AND NASON" were reluctant to suggest flavin involvement in the respiratory chain of Nitrobacter since the 50 % inhibition concentration was 1.0 mM. The strong inhibition which we observed and its reversal by FAD seem to point definitely to the presence of a flavin in the nitrite oxidase system. The results of the spectrophotometric study taken together provide evidence for the occurrence of two cytochromes of type a, although no one aspect of the data may be considered conclusive. The shoulder on the 594-m,u band at 609 m,u was resolved into two clearly different peaks in low-temperature spectra (see Fig. 2), presumably indicating a mixture of a 1 and a cytochromes. The asymmetry of the y band of the at cytochrome at 439 m,u may have been caused by the y band of a at 450 m,u. If al only is complexed with CO, the disappearance of its y band should allow the appearance of the a y band at 450 mf/, since CO bubbling caused an increase in the per cent reduction of cytochromes c and a. The y band at 450 m,u was indeed observed under these conditions and the fact that the depth of the trough at 439 m,u always was smaller than the height of the peak at this wavelength, seems a further argument for the resolution afthe y peak into its two components a 1 and a by CO treatment. The disadvantage of CO camplexing of the terminal oxidase in a cell-free extract is that the y CO band of the cytochrome al, which presumably would have been found at 427 m,u (ref. 12), cannot be separated from the y peak of cytochrome cat 419 m,u. Complexing cytochrome a l by CO should also lead to the extinction of its a band at 594 mj), and a trough should have appeared at this wavelength in the CO difference spectrum: this was in fact observed. Unfortunately the observations on intact cell suspensions are limited to the Soret region and their accuracy is poor even there. Nevertheless the results obtained are in accord with the observations on cell-free extracts with a trough at 440 m,u and a poorly defined peak at 426 mf/,which could be due to the y band of the CO complex of cytochrome al' The composition of the cytochrome chain may thus be summarized as in Table TABLE IV COMPARATIVE SPECTROPHOTOMETRIC VALUES OF NITROBACTER CYTOCHROMES

Wavelength of the maxima of extinction differences between the oxidized and reduced forms of the cytochromes of types a and o,

Author

This paper

Cytochromes a

Cytochromes c

eta

Uy

oa

cp

Cy

609

450

554 55 1 55° 552

523 5 20-525 520

4 19

LEES AND SIM1'SON 1 ALEEM AND NASON' ZAVARZIN"

594 5 89 5 86-59 0 59 2

Biochim: Biopbys. Acta, II3 (1966) 41-50

439 43 8

'P5

MECHANISM OF NITRITE OXIDATION BY NITROBACTER

49

IV, where we have also given the results from other authors on this subject. The absence of a cytochrome of type b seems thus confirmed. The bands for the c-type cytochrome of Nitrobacter at 554, 523 and 419 mj}, are very close to those found by VERNON 13 at 553, 523 and 419 mp,for a Pseudomonas. ORLAND01<1 found a cytochrome of type c in Rhodopseudomonas with maxima at 553, 523 and 418 mp,. The somewhat high wavelength of the a band is characteristic of bacterial cytochrome of type c. Direct spectrophotometric evidence of a flavin involvement in Nitrobacter respiratory chain is difficult to obtain. The pronounced absorption minimum at 465 mj}, in difference spectra obtained after S20~- reduction is quite likely due to the bleaching of flavin components present in cells and cell-free extracts. Reducing with N0 2- causes a much less important minimum at the same wavelength. If a flavin is involved in the nitrite oxidation chain, this implies that the process has to be pulled over a thermodynamically unfavorable step. Even if it is admitted that the combination of the flavin with the apoenzyme raises its potential to about o V, there still remains a large unfavorable difference between this value and that of the N0 2--N0 3 - system (about 004 V). The equilibrium concentration of the reduced flavin will be extremely low and it is quite likely that at the steady state it will still be low, due to this unfavorable thermodynamic relationship. This may account for the fairly low cytochrome turnover in Nitrobacter even though the cytochrome concentrations are higher than those usually found in bacteria. These considerations may be put on a quantitative basis by using CHANCE'S16 figures of merit. Definitions of these figures are recalled in Table V, where some data TABLE V ES1:lMATED QUANTITA1:lVE ASPECTS OF NITROBACTER RESPIRATORY CHAIN

Difference spectra were recorded on intact cell suspensions. The absorbance of the suspensions at m!1 varied from 1.4 to 1.6.

500

Cytochrome a concentration: LJA (Soret peak) 91 000 X mg cell N

IO' 10- 0-20' 10-0

Cytochrome c concentration: LlA (a - peak) 19 000 X mg cell N J{

=

K.1 =

25' 10-9- 45 ' 10-9 moles per mg N

O 2 upta~~J,umoles . sec-l)

A at

1

500

moles per mg N

0.62

m""

O 2 uptake (!Imoles .sec-I) LlA main Soret peak

20

Turnover number = 4' K.· 91' 10-3 equiv- cytochrome-vsec- 1

regarding those and other parameters have been collected after their calculation from spectrophotometric data such as those presented in the first part, using the molar absorption coefficients which have been given by CHANCEI0. It is seen, on the one hand, that the substrate respiration rate as expressed by the parameter ](1 is in the range of values which have been quoted by S:l.UTH17,18 for Biochim, Biophys. Acta, II3 (1966) 41-50

50

A. VAN GOOL, H. LAUDELOUT

other bacteria. Since, on the other hand, the cytochrome. concentrations are fairly high, this must necessarily result in a fairly low turnover number. The relative high cytochrome concentration in Nitrobacter may thus be considered as an adaptation to the thermodynamically unfavorable first step in the respiratory chain. It is obvious that if the equilibrium concentration of the reaction product of one step is very low, one way of ensuring its rapid removal from the system under steady-state conditions consists of increasing the concentration of the carrier in valved in the following step.

ACRNOWLEDGEMENTS

The authors are greatly indebted to Dr, B. CHANCE for the low-temperature spectrum of Fig. 2. This investigation represents part of a programme supported by the Institut pour la Recherche dans l'Tndustrie et l'Agriculture. REFERENCES I H, LEES AND J. R. SIMPSON, Biochem, ]., 65 (1957) 297, W. D. BUTT AND H. LEES, Nature, 188 (1960) 147. 3 G. A. ZAVARZIN, Miltrobiologiya, 27 (195 8) 679. 4 W. S. SILVER, Soil Sci. Soc. Am. Proc., 25 (1961) 197, 5 lVI, 1. H. ALEEM AND A. NASON, Biochem. Biopliys. Res. Commltn., I (1959) 323, 6 H. LEES, Bacteriol. Rev" 26 (1962) 165, 7 B. BOON AND E, LAUDELOUT, Biochem, j., 79 (1960) 39, 8 M. DIXON, Biochem, I-. 55 (1953) 170. 9 lVI. I, E, ALEEM AND A. NASON, Proc. Nail. Acad, Sci. U,S., 46 (1960) 763. 10 E, ENGEL, E. KRICK AND 1. FRIEDRICHSEN, Arch. Mikrobiol., 21 (1954) 96. II 0, MEYERHOF, Arch, Ges, Physiol., 165 (1916) 229. 12 B. CHANCE, J. Biol. cu-«; 202 (1953) 383. 13 L. P. VERNON, J. Bioi. Chem., 222 (1956) 1045. 14 J. A. ORLANDO, Bioohim ; Biophys, Acta, 57 (19 6 2) 373· IS B. CHANCE, ]. Bioi. Cheni., 197 (195 2 ) 5 6 3. 16 B. CHANCE, [, Bioi. Chem., 202 (1953) 407. 17 L. SMITH, Bacteriol. Rev" 18 (rg54) 106. 18 L. SMITH, Bacteria, 2 (Ig61) 386. 2

Biocbim, Biopliys, Acta, II3 (Ig66) 4 1- 5 0