The metabolism of Acetobacter peroxidans

VOL. 21 (1956)

THE

BIOCH1MICAET BIOPHYSICAACTA

METABOLISM OF A C E T O B A C T E R

335

PEROXIDANS*

I. O X I D A T I V E ENZYMES by S T U A R T W. T A N E N B A U M

Department o[ Microbiology, College o/ Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.)

Acetobacter peroxidans was isolated by VISSER 'T HOOFT1 who determined its outstanding physiological characteristics as lack of catalase activity, failure to produce acid from glucose, and growth in Hoyer's medium. WlELAND AND PISTOR2,a investigated the oxidation of ethanol by A. peroxidans using molecular oxygen and hydrogen peroxide as hydrogen acceptors and showed that acetaldehyde is the primary product of this reaction. They also established that the carbohydrates and related compounds, which are commonly attacked by other members of the Acetobacter spp., were not susceptible to oxidation by suspensions of A. peroxidans. A chromatographic study of the culture media of the various acetic acid bacteria grown in yeastextract sugar solution by FRATEUR et al. 4 demonstrated in the case of A. peroxidans that no reducing compounds other than the initial substrate are detectable. Apart from these observations the metabolic activities of this potentially interesting microorganism have been largely neglected. This paper reports the results of a reinvestigation and a general survey of the oxidative activities of A. peroxidans. MATERIALS AND METHODS

Acetobacter peroxidans strain N C I B 8618 was used. Preliminary e x p e r i m e n t s h a v e s h o w n t h a t the metabolic b e h a v i o r of strain ATCC 838 is largely similar. The microorganism was maintained at 3 °0 C on agar slants of a modified (FRATEUR5) H o y e r ' s ~ % ethanol m e d i u m of the following composition: per 1 of t a p water, Na acetate, 1 g; (NH4)2SO4, I g; K H 2 P O 4, 0.9 g; K 2 H P O 4, o.i g; MgSO 4, 0.25 g; and 0. 5 ml of i % FeC1 a. For g r o w t h in liquid media the Na acetate was omitted. S t a r t e r cultures were m a d e b y inoculating the washings from a fresh slant into 500 ml of the above mixture. Agitation was achieved by stirring magnetically at 3 °° C and m a x i m u m t u r b i d i t y was observed at the end of 48 hours. The s t a r t e r culture was added to ~5 1 of sterile m e d i u m contained in a carboy, and the entire c o n t e n t s were vigorously aerated for 48 to 72 hours. The cells were collected by centrifugation and washed twice with t a p water. Dried cell p r e p a r a t i o n s were made by direct lyophilization of the w a t e r - w a s h e d cells. Cell-free e x t r a c t s were obtained b y sonic disintegration of suspensions in a R a y t h e o n 9 kc oscillator for 17 minutes, followed b y centrifugation at io,ooo r.p.m, for one h o u r in the cold. The crude e x t r a c t was stored at - - 7 o° C. Despite these precautions, m a n y of the oxidative activities p r e s e n t in the fresh crude e x t r a c t s decreased rapidly within a week. I t should be noted that, in general, the e n z y m e s found in the cell-free e x t r a c t s of ,4. peroxidans h a v e t h u s far p r o v e d to be quite unstable, regardless of the m e t h o d of p r e p a r a t i o n . The addition of sulfhydryl agents, nicotinamide or o t h e r cofactors comm o n l y employed as stabilizing agents, has not enhanced the stability of these extracts. \Vhole * This investigation was s u p p o r t e d by a research g r a n t (E-767) from the National Microbiological I n s t i t u t e of the National I n s t i t u t e s of Health, Public H e a l t h Service.

References p. 342.

336

s . w . TANENBAUM

VOL. 21 (1956)

cells maintained most of their oxidative capacities for several days when kept at icebox temperature. Where indicated in the text, "yeast-extract grown cells" refers to suspensions of ,4. peroxidans grown in a o.25 % yeast extract (Difco), 2 % ethanol medium in shake culture. Microscopic examination of cultures in the log phase revealed short rods, often joined in pairs, with a morphology inore closely resembling the catalase-negative dcelobacler paradoxum shown by I~RATEUR~. Specimens from aerated cultures invariably exhibited organisms with marked motility, which ceased after several minutes on the slide. Besides morphological examination, each batch of cells was tested for failure to oxidize glucose in the Warburg apparatus and for lack of catalase activity. Protein was determined by the Folin-Ciocalteau method (KABAT AND MAYER6), using bovine serum globulin as a standard, and phosphate by a modification of the procedure of LowRY AND I,OPEZ7. DPN* and TPN were commercial preparations, antimycin A was obtained from the \Visconsin Alumni Research Foundation, cytochrome c from Sigma Chemical Co., and thiomalic acid from the Evans Cosmetic Corporation. Oxalacetic acid was kindly provided by Dr. A. KRASXA, Na glyoxylate by Dr. D. B. SPRINSON, and methyl peroxide by Dr. BRVrTEN CHANCE. Na glycollate was prepared by the saponification of the ethyl ester (Eastman). RESULTS

Oxidation o~ alcohols

F r e s h l y h a r v e s t e d w a s h e d cells (Fig. I), cells a g e d i n t h e cold, a n d d r i e d cell p r e p a r a t i o n s , e a c h o x i d i z e d e t h a n o l t o CO 2 a n d w a t e r . W i t h w h o l e cells, I . I O 4 M 2, 4d i n i t r o p h e n o l w a s w i t h o u t effect u p o n t h e c o u r s e of e t h a n o l o x i d a t i o n , w h e r e a s cyanide and azide (I.IO-3M) completely inhibited oxidative activity. During e t h a n o l o x i d a t i o n b y f r e s h cell s u s p e n s i o n s , c o n s i d e r a b l e a n d v a r i a b l e a s s i m i l a t i o n t o o k place. W i t h 2 4 - h o u r a g e d cells, h o w e v e r , t h e R . Q . a p p r o a c h e d 0.5 a n d t h e

500

500

- 20 # M

o~

PtOH + I 0 "4 M nNp

~

400

~_

---

~ C02

300

.--e

.Ac

w#M //

1/

2oc

--

00i O"

10

20

200 ~

-o2

/

i

,

30

40

i

/

~ //;,.

i

5'0 60 70 80 Time in minutec

Fig. i. Ethanol and acetate oxidation by freshly harvested water-washed cells of .4. peroxidans. \Varburg vessels contained 15 mg (dry weight) of cells in 2.5 ml phosphate buffer pH 5-9. l o / t m o l e s of substrates added. DNP = 2, 4dinitrophenol. Temperature 3 °° C.

10

CHaCH2OH + 202 ~

20

30 40 Time in minutes

Fig. 2. Ethanol and acetate oxidation by cells which were aged 24 hours in tile cold. 1)rotocols as given under Fig. 1.

r e l a t i o n s h i p of t h e CO 2 e v o l v e d t o 0,, c o n s u m e d following equation:

---

(Fig. 2) w a s i n a c c o r d w i t h t h e

CO 2 + (CH20) + 2H20

(l)

* Abbreviations used: DPN, diphosphopyridine nucleotide, coenzyme I; TPN, triphosphopyridine nucleotide, coenzyme II; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; CoA, coenzyme A; R.Q., respiratory quotient; O.D., optical density; ATP, adenosine triphosphate ; il ), inorganic phosphate.

Re/ere~ces p..H~"

VOl.. 21 (I956)

METABOI.ISM OV Acetobacter peroxidans I

337

Butanol oxidation stopped at butyric acid. The inability of this microorganism to attack the latter substance was confirmed in additional experiments. Beside glucose and fructose, glycerol, glycollic and glyoxylic acids were indifferent substrates to suspensions of A. peroxidans. Cell-free extracts usually oxidized ethanol and acetaldehyde. Treatment of these extracts with up to 20 vol per cent of Dowex-I as recommended by STADTMaN et al. a for removal of CoA did not diminish their ability to oxidize either snbstrate, when rates were calculated on the basis of the protein content. This indicated that CoA is probably not involved in the conversion of the aldehyde to acetate by this particular species. The conversion of ethanol to acetaldehyde was unaffected when washed cells in phthalate buffer were exposed to i. IO a M arsenate, whereas acetaldehyde oxidation was completely arrested. Although these results imply a role for phosphate during aldehyde oxidation, acetyl phosphate itself is not further metabolized by A. peroxidans. The oxidation of ethanol and of acetaldehyde by cells or by cell-free preparations was not prevented by concentrations of antimycin A (POTTER AND REIF 9) as high as 20 ~g per Warburg flask. This establishes the existence of yet another antimycin A-resistant electron transport chain in microorganisms.

Alcohol and ahtehyde dehydrogenases The oxidation of ethanol and acetaldehyde by crude cell-free preparations were T P N specific (Fig. 3). The substitution of DPN ~L1.3 r for T P N did not result in any increase in tO# MOLES ~,t~50H ~ c ~ "*'°--'~ O.Sll#MOLES TPN optical density at 34o m/x. Again, TPN INS?If PROTEIN reduction could still be demonstrated ._.._-I o25o .~. during acetaldehyde oxidation in Dowex-i 0.225 o :~ o.91Io uI/O~..ESCH3CHO treated extracts. 0.200 ~ 0.8 0.24# MOLES TPN

,of I

TPNH-oxidase and peroxidase

0.7 K

o 0.6

[~N

0.175 I 0.150 "~

When cell-free extracts were examined O.la5 0.5 spectrophotometrically several hours after 0.100 ~" 0.4 0.075 o their preparation for TPN reduction with 0.3 0.050 formate, pyruvate or oxalacetic acid, the O.2 0.025 maximum O.D, attained at 34o m/x fell O.1 off rapidly in a few minutes. Since this 3 b 9 12 15 18 21 24 27 30 Time in minute~ diminution in O.D. was not prevented by Fig. 3- R e d u c t i o n of T P N b y a l c ohol a n d io -a 3I cyanide, it is believed that these a l d e h y d e d e h y d r o g e n a s e s in cell-free expreliminary observations are a measure t r a c t s . O p t i c a l d e n s i t i e s on left o r d i n a t e refer t o e t h a n o l o x i d a t i o n , t h o s e on r i g h t of a flavin-linked oxidase which by-passes ordinate to acetaldehyde oxidation. the cytochrome system. Another manifestation of this TPNH-oxidase or diaphorase activity is the demonstration (see below) of T P N H oxidation with indophenol. When T P N H was generated in these extracts by means of the ethanol dehydrogenase system, the subsequent addition of H202 resulted in a peroxidation of the reduced coenzyme I I (Table I). This oxidation and that of reduced eytochrome c by the peroxidase-peroxide system are probably analogous to the oxidative mechanisms in yeast, first described by CI~aNCE1°. Re/crevices p. 34 e.

338

s . w . TANENBAUM

VOL. 21 (I956)

TABLE I PEROXIDATION

ENZYMIC

OF

TPNH

AND

OF REDUCED

T/me

CYTOCHROME

C IN

O . D . ~4o m p • ~o ~ Additions

o

A

B

208

248

331 75 ° 807

37 ° 820 85o (822) 805 797

l

5 [o 1%

H202"

(780) **

74 ° 670 582 337

5 IO 20

O . D . 5 5 o m t l • ~(,a C

o. I ml EtOH

O.I ml EtOH (to#M)

o.[ ml

EXTRACTS

Additions

rain

I

CELL-FREE

O.1 ml H202

782 737

485 590 625 (605) 409 489 479 46I

Protocols: Cuvettes A and B; 0.5 ml cell-free preparation (i.16 mg protein), 2.2 ml phosphate pH 5.9, o.2 ml (0.25/*inole) TPN. Cuvette C; 0. 5 ml cell-free preparation (1.16 mg protein), 2.2 ml phosphate buffer, o. t 4 m[ (o.i 75 tanole) TPN, ].o mg cytochrome c. Cuvette B received o.[ ml water instead of H202. ** Figures in parentheses represent calculated O.D. on basis of dilution.

A celate oxidation A c e t a t e was a t t a c k e d b y b o t h fresh a n d a g e d cells. T h e cell-free p r e p a r a t i o n s were d e v o i d of e n z y m i c a c t i v i t y a g a i n s t this s u b s t r a t e , w h e r e a s u n c e n t r i f u g e d s o n i c a l l y d i s r u p t e d p a r t i c l e s m a i n t a i n e d this o x i d a t i v e a b i l i t y . T h e R . Q . for a c e t a t e o x i d a t i o n c o n s i s t e n t l y a p p r o a c h e d I, a n d e x p e r i m e n t s w i t h 24-hour a g e d cells (Fig. 2) a g a i n indicated the conversion: CHaCOOH + O 5---+ CO 2 + (CH20) + H20 Acetate ~°° /]l g

/

oxidation

was a l m o s t

02

~

c02

40o~ g*s/,Mt-

300l

S

IOjuMAc

loF~ P //

dIOjuMBuOHt-KOH 200~ ~'~'O~uM BuO' 5juMPy IOjuMFORM

'°Oil~~~ .

ji

30 6o 0o le0 ~o ~80 21o Time in

minu'i'es

Fig. 4- Oxidation of formate (form), pyruvate (py), butanol (BuOH), and acetate (Ac) by intact fresh cells, Protocols as under Fig. 2. IAC, iodoacetate (~o 4M).

(2)

c o m p l e t e l y i n h i b i t e d b y 1 . 1 0 _4 M d i n i t r o p h e n o l (Fig. I) as well as b y a s i m i l a r c o n c e n t r a t i o n of i o d o a c e t a t e , in e x p e r i m e n t s w i t h w h o l e cells. Cells of A. peroxidans g r o w n in y e a s t e x t r a c t e x h i b i t e d no d e m o n s t r a b l e differences f r o m cells g r o w n in H o y e r ' s m e d i u m as far as o x i d a t i o n of e t h a n o l , acetaldehyde and acetate were concerned.

Pyruvate and/ormate oxidations P y r u v i c a c i d was r a p i d l y o x i d a t i v e l y d e c a r b o x y l a t e d b y s u s p e n s i o n s of A. peroxidans (Fig. 4). Q u a n t i t a t i v e t e s t s for a c e t o i n or d i a c e t y l in t h e reaction medium were negative. Spectrophotom e t r i c e x a m i n a t i o n of this r e a c t i o n in t h e cellfree s y s t e m i n d i c a t e d t h a t t h e r e a c t i o n c o u l d be l i n k e d o n l y to T P N r e d u c t i o n . L a c t a t e beh a y e d in t h e s a m e m a n n e r as p y r u v a t e . F o r m a t e was q u a n t i t a t i v e l y o x i d i z e d to CO 2 a n d w a t e r a, as s h o w n in Fig. 4 a c c o r d i n g to HCOOH + ~'z O2--+ CO2 + H20-

(3)

A g a i n , f o r m i c d e h y d r o g e n a s e in cell-free e x t r a c t s c o u l d be e i t h e r T P N or D P N linked.

Re/erences p. 342.

VOL. 21 (1956)

METABOLISM OF Acetobacter peroxidans I

339

Tricarboxylic acid cycle intermediates The oxidative decarboxylation of the dicarboxylic acids involved in the Krebs cycle is depicted in Fig. 5. Succinate, oxalacetate, malate and fumarate were attacked without a lag period. The oxidation of the latter two substrates was in contrast to the earlier observations ,r_.OOAA 600 it. of WIELAND AND PISTOR3, who listed these among / a large number of metabolic intermediates untouched f by the strain of A. peroxidans they employed. Thio- e~l ~00 ~ IIII I ~'eSuc¢ malate (4-io -a M) inhibited malate oxidation by 0 intact cells. In sonically disrupted suspensions the 400 I I oxidation of succinate was not inhibited by 2" IO-aM / t ~ S ucc malonate, whereas pyrophosphate (2.io-3M) and // / iodoacetate (4" lO-3 M) reduced oxygen consumption 300 Fum by 75 %,, I1// / ~ - - . - o Ma~ Citrate, isoeitrate and a-ketoglutarate were not / / 200 II /, /'I// // /' I oxidized by suspensions of A. peroxidam ; nor were these substances attacked by the sonically disrupted I M,V/ - - 02 loo / / / ~ / - - - co2 non-centrifuged mixtures which oxidized acetate. ,y/ Furthermore, they were not oxidized by the cell-free I~'01+ THIO preparations. Attempts to initiate a-ketoglutarate 20 4 0 60 80 100 120 140 or tricarboxylic acid oxidation by the addition of Time in minute~ sparker amounts of dicarboxylic acids or by the Fig. 5. Oxidative decarboxylation addition of such cofactors as CoA, TPN, DPN, lipoic of the dicarboxylic acids by cell suspensions. Protocols as given acid or manganous ion, were unsuccessful. Extracts under Fig. i; substrate concentration io ymoles. OAA, oxalacetic which contained active pyridine nucleotide-linked acid; Succ, succinic acid; Fum, oxalacetic, malic, lactic, alcohol, and aldehyde de- fumaric acid; Mal, malic acid; hydrogenases failed to reduce the coenzyme in the THIO, thiomalic acid (Io ymoles). presence of isocitric acid.

/ t(// ~o~

TPN-cytochrome c reductase A fresh cell suspension was made by centrifuging the contents of a shake flask, washing twice with tap water, and resuspending in phosphate buffer p H 5.9. It was examined immediately with a Zeiss-Winkel hand spectroscope. Although this sample had no absorption in the green region, the addition of dithionite or of ethanol immediately resulted in a single visible absorption band at 553 ± 5 m/x. Formate also acted as a hydrogen donor to the bacterial cytochrome. These observations confirm those of WIELAND AND PISTOR2, who observed cytochrome bands in A. peroxidans upon addition of strong reducing agents, and are supplemented by the more precise spectrophotometric observations of CHIN11. When 20/xmoles of ethanol were added to a freshly prepared cell-free extract of A. peroxidans in a Beckmann cuvette, there was a distinct increase in optical density at 55 ° m/x; this was rapidly increased further by the subsequent addition of o.I/xmole TPN. Moreover, the reduction of mammalian cytochrome c could be demonstrated b y utilizing the ethanol-TPNH generating system found in A. peroxidans (Fig. 6). Again, this reaction was observed only in very freshly prepared extracts. When these were kept at - - 7 o ° C for 24 hours no cytochrome ¢ reduction could be detected, although T P N H production remained unimpaired. Re/erences p. 342. 22

34 °

s . w . TANENBAUM

VOL. 21 (1950)

Cytochrome oxidase and peroxidase Attempts to demonstrate cytochrome c oxidase manometrically after adding hydroquinone, ascorbic acid or p-phenylenediamine with cytochrome to the cell-free extracts were unsuccessful. Likewise, there was no decrease in O.D. at 55o m/~ in experiments whereby reduced cytochrome c was formed via the e t h a n o l - T P N H cytochrome reductase system when the cuvettes were shaken with air in the absence of cyanide. SMITH12 has tested suspensions of disrupted cells from eight species of bacteria containing cytochromes for cytochrome c oxidase activity with essentially negative results and has pointed out that the respiratory enzymes at this portion of the chain in several acetic acid bacteria differ from those of heart muscle and yeast. The failure to find cytochrome c oxidase in these experiments is not entirely unexpected. CHINn has presented preliminary evidence to show that cytochrome a 2 is the autoxidizable component in A. peroxidans. The addition of H20 2 to reduced cytochrome c formed in the cell-free extracts resulted in a decrease in O.D. at 550 m/~, demonstrating the presence of cytochrome c peroxidase in A. peroxidans (Table I). The increase in density after an initial decrease with H202 may indicate an equilibrium attainment between the reduced and oxidized forms of the enzyme in the presence of excess ethanol, T P N and H20 2.

Indophenol and/erricyanide reductase Experiments in anaerobic Thunberg tubes with cell-free extracts at pH 6 showed that 2,6-dichloroindophenol was rapidly reduced by the ethanol-TPNH system. This enzymic activity can be taken as presumptive evidence for the presence of diaphorase activity in A. peroxidans. Methylene blue was a sluggish hydrogen acceptor compared to indophenol under these conditions. In addition, the indophenol reductase reaction was followed spectrophotometrically at two hydrogen ion concentrations (Fig. 7)In separate experiments there was no evidence for non-enzymic oxidation of T P N H by indophenoP 3,14. Ferricyanide also acted as a hydrogen acceptor for T P N H (Fig. 8). Indophenol reductase was demonstrable in aged extracts long after cytochrome c reductase activity had been abolished, suggesting again that an independent diaphorase was present rather than cytochrome e reductase manifesting diaphorase activity.

Peroxidase Although A. peroxidans lacks catalase, WIELAND AND PISTOR2 demonstrated that this microorganism can catalyze the analogous hydroperoxidase reaction with ethanol, and the hydroperoxidation of T P N H and reduced cytochrome c have been described earlier in this paper. Peroxidase activity was also demonstrated by the modified purpurogallin test of CHENG15 and the results of a typical experiment are shown in Fig. 6. Suitable corrections for non-enzymic oxidation of the substrate by H202 have been made. Monomethylhydroperoxide cannot be substituted for H202 in this enzymic peroxidation of pyrogallol.

Relation to oxidative phosphorylation In Table I I are given the protocols ot some experiments attempting to ascertain whether the esterification of inorganic phosphate at the substrate level attends ethanol, acetaldehyde or suecinate oxidation. The reactions were carried out in the Warburg apparatus in phthalate buffer, pH 5.9. It can be estimated from the amounts of oxygen consumed that ethanol and acetaldehyde were taken almost completely to Re/erem es p. 342.

VOL. 2 1 (1956)

METABOLISM OF

0.400 ::L E

Acetobacter peroxidans

I

341

0.400 t :eL E

~n

~ 0.30o

0.300 d

0.200

0.200

0.100

0.100

2

4

6

i 10 12 14 16 Time in minutes

8

Fig. 6. C u r v e I. P e r o x i d a s e a c t i v i t y in cell-free e x t r a c t s ( / ~ O . D . 425 m/~). C u v e t t e c o n t a i n e d 0. 5 m l c r u d e e n z y m e (0.764 m g protein), o.I ml fresh l y p r e p a r e d 5 % p y r o g a l l o l , o.[ ml i % H20~, a n d p h o s p h a t e buffer p H 5.9 to a final v o l u m e of 3.0 mi. C u r v e 2. R e d u c t i o n of c y t o c h r o m e c b y t h e e t h a n o l T P N H s y s t e m (/~ O.D. 55 ° m/,). C u v e t t e c o n t a i n e d 0. 5 ml e n z y m e e x t r a c t , 0.2 m l E t O H (20/ ,mol e s ), o.i ml T P N (0.23 /~mole) a n d i .o m g c y t o c h r o m e c, in phosp h a t e buffer p H 5.0 to final v o l u n l e of 3.0 ml.

0.400 0.70O

::L E

2

0.600 ::L. i E 0.500

.

.

.

.

-

0.300 w

d d

g

~o c~ d

0.400

0.200

0.300 0.200

0.100

0.100 i 1

2

3

4

4

5 6 7 8 Time in minutes

8

1'~ 1;

2;

2,

Time in minutes

Fig. 8. F e r r i c y a n i d e o x i d a t i o n of T P N H in cellfree e x t r a c t s . C u v e t t e s c o n t a i n e d (Curve i) o.2 ml e n z y m e (o.149 m g protein), o.2 ml (o.i /~mole) f e r r i c y a n i d e , o.i m l T P N (o.23 #mol e ), 2. 3 ml p h o s p h a t e p H 5.9. R e a c t i o n s t a r t e d b y t h e a d d i t i o n cf 0.2 m l (I0/~moles) of e t h a n o l . In Curve 2 TPN was omitted.

Fig. 7. R e d u c t i o n of i n d o p h e n o l in cell-free ext r a c t s . C u v e t t e s c o n t a i n e d o.1 ml cell-free p r e p a r a t i o n (o.o76 m g protein), o.2 m l dye (stock c o n c e n t r a t i o n o.2 m g / m l ) , o.2 m l aceta l d e h y d e (2oo/~moles) a n d p h o s p h a t e buffer to 3.o ml v o l u m e . C u r v e i, p H 6.o; C u r v e 3, p H 7.o, C u r v e 2, no a c e t a l d e h y d e added, endog enous r e d u c t i o n .

TABLE

II

ATTEMPT TO DEMONSTRATE OXIDATIVE PHOSPHORYLATION St,stem

Cells Cells "&Thole s o n i c a t e Cell-free Cell-free Cell-free

Substrate

ethanol acetaldehyde succinate ethanol acetaldehyde --

Amount #moles

2 oo 200 ioo 200 200 --

O~ uptake I~atoms

z~ iP I~moles

182 92 91 116 68 o

o o - - i .o + 1.65 + 1.o8 o*

W a r b u r g vessels c o n t a i n e d t o t a l v o l u m e of 2.5 m l ; p h t h a l a t e buffer p H 5.9 w i t h 2 0 / * m o l e s i P i n i t i a l l y added. D r y w e i g h t of cells, 16 mg. Cell-free e x t r a c t s c o n t a i n e d 3.05 m g p r o t e i n . * B l a n k v a l u e s for i P in t h e s e cell-free c o n t r o l s r a n g e d from 0.375 to o . 5 5 o / , m o l e s . A T P a s e a c t i v i t y w a s negligible in these p r e p a r a t i o n s .

342

s . w . TANENBAUM

VOL. 21 (1956)

the stage of acetate by the cell suspensions, succinate to the oxidation level of oxaloacetate by the uncentrifuged sonically disrupted cells, and ethanol to acetaldehyde by the cell-free extract. It was not necessary to add fluoride, since special experiments showed that ATPase activity is negligible in the disrupted and cell-free preparations. In spite of the large consumption of 0 3, no iP disappeared. Further experiments with cell-free extracts oxidizing the above substrates in the presence of glucose, ADP, and hexokinase again failed to reveal any iP uptake. One cannot rule out lack of high energy phosphate bond formation during these oxidative transformations in A. peroxidans, however, unless the appropriate radioactive phosphorus experiments concur with the analytical evidence presented at this time. DISCUSSION

The results obtained here reveal that a cytochrome type of respiration is probably operating in the catalase-lacking A. peroxidans in contrast to a direct flavoprotein to oxygen pathway. This conclusion is based upon the older observations of cyanide and azide inhibitions of respiration, confirmed here, and upon the demonstration of TPN-linked dehydrogenases and TPNH-cytochrome c reductase in cell-free extracts. The extent to which H202 peroxidations of T P N H or of reduced bacterial cytochrome, also seen in these extracts, operate in the intact functioning cells can probably best be ascertained by the techniques developed by CHANCETM. It would appear from the negative results reported above that terminal acetate oxidation takes place in A. peroxidans by a dicarboxylic rather than by a tricarboxylic acid cycle. The pitfalls involved in making a decision on the basis of the type of data obtained have been discussed by SWIM AND KRAMPITZTM. A definitive answer to whether A. peroxidans possesses the usual Krebs cycle must await application of the suitable tracer methodology developed by these investigators. SUMMARY S e v e r a l o x i d a t i v e e n z y m e s of t h e c a t a l a s e - n e g a t i v e b a c t e r i u m Acetobacter peroxidans h a v e been i n v e s t i g a t e d . Alcohol a n d a c e t a l d e h y d e d e h y d r o g e n a s e s are TPN-specific. T P N H oxidase, T P N H l i n k e d c y t o c h r o m e c r e d u c t a s e , d i a p h o r a s e , p e r o x i d a s e , a n d t h e H 2 0 ~ p e r o x i d a t i o n s of r e d u c e d T P N a n d of r e d u c e d c y t o c h r o m e can be d e m o n s t r a t e d in cell-free e x t r a c t s . The e n t i r e e l e c t ron t r a n s p o r t c h a i n is a n t i m y c i n A i n s e n s i t i v e . The c o n v e r s i o n of a c e t a l d e h y d e t o a c e t a t e is not CoA d e p e n d e n t . I t a p p e a r s as if t h e u s u a l t r i c a r b o x y l i c acid cycle is i n o p e r a t i v e d u r i n g a c e t a t e o x i d a t i o n by this microorganism. REFERENCES 1 F. VlSSER 'T HOOFT, Dissertation, Delft, I925. 2 H. WIELAND AND H. J. PISTOR, Ann. Chem., Justus Liebigs, 522 (1936) 116. 3 H. \¥IELAND AND H. J. PISTON, Ann. Chem., Justus Liebigs, 535 (I938) 205. 4 j . FRATEUR, P. SIMONART AND T. COULON, Antonie van Leeuwenhoeh J. Microbiol. Serol., 20 field, (1954) i i i . 5 j . FRATEUR, La cellule, 53 (195o) 287. E. A. KABAT AND M. M. MAYER, Experimental Immunochemistry, Charles. C. T h o m a s , Springfield, II1., 1948, p. 32I. : O. H. LowRY AND J. A. LOPEZ, J. Biol. Chem., 162 (1946 ) 421. 8 E. R. STADTMAN, G. D. NOVELLI AND F. LIPMANN, J. Biol. Chem., 191 (1951) 365 . 9 V. R. POTTER AND A. E. REIF, J. Biol. Chem., 194 (1952) 28710 B. CHANCE, Nature, 169 (I952) 215. 11 C. I-I. CHIN, 2nd Intern. Congr. Biochem. Abstr. o/Communs., (1952) 277. is L. SMITH, Baeteriol. Revs., 18 (1954) lO6. 13 E. HAAS, J. Biol. Chem., 148 (1943) 481. 1, M. I. DOLIN, Arch. Biochem. Biophys., 55 (195.5) 415 . 1~ S. CHENG, Plant Physiol., 29 (1954) 458. 16 H. E. SW'IM AND L. KRAMPITZ, J. Bacteriol., 67 (1954) 419.

Received October I7th, 1955