Enzymic sulphide oxidation by Thiobacillus concretivorus

114 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26139

ENZYMIC S U L P H I D E OXIDATION BY THIOBACILLUS CONCRETIVORUS

D. J. W. M O R I A R T Y AND D. J. D. NICHOLAS

Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide, Adelaide (Australia)

(Received December i2th , 1968)

SUMMARY

I. The oxidation of sulphide by Thiobacillus concretivorus, Thiobacillus thiooxidans and Thiobacillus thioparus is an enzymic process associated with cell mem- branes.

2. The first reaction is an oxidative one involving electron transfer through the cytochrome system to oxygen. I t is inhibited by sodium diethyldithiocarbamate and Tris-HC1, but not by CO or sodium azide.

3. The Km of sulphide oxidation is 2. IO -~ M. 4. Conjugated sulphur compounds with distinctive spectral properties are sub-

sequently formed. These may be polysulphides. This step is inhibited by CO. 5. A native cytochrome c which has been purified I2o-fold from T. concretivorus

is involved in the formation of the polysulphur intermediates. CO binds to this cytochrome, and inhibits the production of the polysulphur compounds which absorb around 320 m~.

6. A role for copper and the formation of a bound intermediate is proposed. 7. A heat-stable factor, essential for sulphide oxidation, has been extracted in

acetone from the membrane fraction.

INTRODUCTION

The thiobacilli, a group of chemoautotrophic bacteria, obtain energy for growth by oxidising reduced inorganic sulphur compounds. The oxidation of one molecule of sulphide to sulphate is a process requiring an 8-electron transfer.

The mechanism of sulphide oxidation has not been investigated in detail by previous workers. Elemental sulphur 1,2, thiosulphate and polythionates2, a have been proposed as intermediates in this process. Cell-flee preparations have been shown to oxidise sulphide with a concomitant reduction of cytochrome c4, 5. ADAIR s, however, suggests that sulphide oxidation occurs chemically and that cytochromes are not involved.

In this paper Thiobacillus concretivorus has been studied in some detail and it is shown that sulphide oxidation is an enzymic process for which a mechanism can now be proposed.

Biochim. Biophys. Acta, i84 (1969) 114-123

SULPHIDE OXIDATION BY T. concrelivorus 115

METHODS AND MATERIALS

Culture of organisms A continuous culture metho& has been adapted for growing T. concretivorus

(NCIB 9514). The medium was that of VISHNIAC AND SANTER 2 for T. thiooxidans with the exception that the trace metal solution was diluted io times and the pH was adjusted to 5.6 by using 0.5 g/1 K2HPO 4 and 7.5 g{1 of KH2PO 4. The culture medium was sterilised by passing it through a Millipore filter (0.45 t*). The pH of the inoculum (5 %, v/v) when fully grown, was adjusted from pH 1.5 to 5.5 with 25 % (w/v) K2CO a before inoculation. During growth, the continuous culture was maintained at pH 5.0 and the cell density was kept at about 0.3 g/1 by appropriate adjustment of the flow rate.

Cells were collected at 4 ° in a Sorvall RC-2 refrigerated centrifuge, fitted with a continuous flow head. They were washed in 50 mM phosphate buffer (pH 7.0) and stored as a paste at --15 °.

T. thiooxidans (NCIB 9112) was grown in the same medium, in 4o-1 batches at 3 o°. The culture was sparged with air. After 7 days when the pH had fallen from 5.6 to around 1.5, the cells were collected and stored as for T. concretivorus.

T. thioparus (NCIB 8349) was also grown in 4o-1 batches. The medium was that described by VISHNIAC AND SANTER 2 for T. thioparus, but the trace element solution was diluted IO times. After 7 days growth, during which the pH was periodically adjusted to 7.0 with 25 %(w/v)K2CO3, the cells were collected and stored as above.

Crude extracts of Azotobacter vinelandii were prepared as described previously 8.

Preparation of extracts Cells suspended in 50 mM phosphate buffer (pH 7.0) containing 0.2 mM Na-

EDTA (25%, w/v) were passed twice through a French Pressure Cell at 20000 lb.inch -z at 4 °. The crude homogenate was centrifuged at 20 ooo ×g for 40 min, and the supernatant fraction was used as the crude extract after dialysis for 12 h against 200 vol. of the same buffer. The crude extract was centrifuged at 144 ooo ×g for i h yielding a supernatant fraction and a pellet. The pellet was resuspended in 50 mM phosphate (pH 7.0) containing 0.2 mM Na-EDTA, and this will be referred to as the membrane fraction.

Protein was determined by the method of ITZHAKI AND CJILL 9.

Sulphide measurements Sulphide concentration was continuously monitored by a sulphide electrode

(Orion Research, U.S.A.) connected to a Beckman research pH meter and Goerz Electro Servoscribe recorder. A full-scale deflection of 2oo mV was used, for which the response time was I sec.

To 3 ml of 5o mM phosphate buffer (pH 7.o) containing o.2 mM Na-EDTA in a 25-ml beaker, io mM sodium sulphide was added until the required concentration was reached, as measured by the electrode. The reaction was started by rapidly injecting the enzyme from a syringe into the reaction mixture which was swirled continuously with a magnetic stirrer. All reactions were carried out at 25 °.

The vessel was made anaerobic by sealing with a tightly fitting rubber stopper, through which the electrodes were passed, and flushing with N 2 (free of 02). Syringe

Biochim. Biophys. Acta, 184 (1969) 114-123

116 D. j. w. MORIARTY, D. J. D. NICHOLAS

needles were used for gas inlet and escape, and for the addition of enzyme and sulphide. With the first sulphide addition, a slow reaction occurred until the residual 0 2 was depleted. No further reaction took place until air was readmitted.

The response of the electrode in mV is a log function of sulphide concentration and so the progress curve of the enzyme reaction is exponential (compare Figs. I and 2). The tangent to this curve at the point of addition of enzyme is an estimate of the initial rate in terms of mV per unit time. A close estimate of the molar rate of sulphide oxidation was conveniently calculated by multiplying the mV rate value by the cor- responding initial sulphide concentration.

Oxygen uptake Oxygen uptake was measured polarographically with a Beckman oxygen sensor

(Model 39 065) fitted with an adaptor box (96 260) and a Beckman recorder. The reaction mixture in the vessel contained 4.0 ml of 5o mM phosphate buffer and 0.2 mM Na-EDTA, and 20/~1 each of crude extract (5 mg protein/ml) and N%S (I raM) were injected into it. The sulphide did not affect the electrode during the short reaction time.

S pectr op hotometry A Unicam SP 8oo recording spectrophotometer was used in conjunction with a

scale expander and Goerz Electro Servoscribe recorder to follow the formation of sulphur compounds and to measure changes in cytochrome components.

Determination of copper and iron Dried membrane fractions from the bacterium (25 mg protein) were digested

with I part 18 M H2SO 4 and 3 parts IO M HNO 3 and then diluted to 2.5 ml with distilled water. Blank determinants and standards were treated similarly. Readings were made in a Techtron atomic absorption spectrometer (Model AA4).

Purification of cytochrome c The cytochrome c from the 144 ooo ×g supernatant fraction of T. concretivorus

was precipitated with ammonium sulphate between 60 and 80% saturation. The pre- cipitate, resuspended in I mM phosphate (pH 7.0) was extensively dialysed against the same buffer. I t was then applied to a DEAE-cellulose column (1.5 cm X 15 cm) and eluted with 2 mM phosphate (pH 7.0). The cytochrome fractions were concentrated by dialysis against polyethylene glycol solution. After further dialysis against I mM phosphate (pH 7.0) the cytochrome was applied to a CM-cellulose column (1.5 cm × 15 cm) and eluted with a gradient of 2O-lOO mM phosphate (pH 7.0). The cytochrome collected at around 50 mM phosphate was concentrated as described previously 8. I t was purified i2o-fold relative to the crude extract, based on the absorbance ratio of 280 to 415 m~.

Acetone extraction The membrane fraction was extracted in 9 ° % acetone at --15 °. The acetone-

insoluble material was separated by centrifuging at 20 ooo x g for 15 min and acetone removed from the supernatant fraction by means of a rotary evaporator. The acetone- soluble and the acetone-insoluble fractions were resuspended in 5o mM phosphate (pH 7.0) buffer.


SULPHIDE OXIDATION BY T. concretivorus 117

Chemicals Standard A.R. grade chemicals were made up in double glass-distilled water. A

solution of Na2S was freshly made each day from crystals washed with water and then dried. Mammalian cytochrome c (horse heart type I I I ) was purchased from Sigma Chemical, St. Louis, U.S.A. ; sodium diethyldithiocarbamate from Merck, Darmstadt , Germany; 2,2'-dipyridyl, 8-hydroxyquinoline and o-phenanthroline from British Drug Houses, Poole, England.

RESULTS

Sulphide is rapidly oxidised by whole cells and their extracts, as determined by the sulphide electrode (Fig. I). The reaction occurs in 2 stages. First there is a rapid oxidation, which is dependent on the amounts of sulphide and crude extract added and will be referred to subsequently as Stage I. I t is followed by a slower reaction (Stage 2) as indicated in Fig. i.

15C

10(

g 5(

Stage 2

lid 2aO 3~) 40 Time (see)

150

IO0

~o~

E

2

N

f~

42(i

2 1 2 2

5

5

~O-4

10-5 v 2

0-6"~

10 .7 2

I ~o 8

cx

10 15 Time (see)

Fig. I. Comparison of sulphide and oxygen uptake by crude extracts (see under MATERIALS) a t 25 °. Reaction mixture in each case contained 5 ° mM phosphate buffer (pH 7.o), 0.2 mM EDTA and 5 mg protein in total vol. of 3.2 ml. At zero t ime 50/~1 of io mM Na2S was added. - - , sulphide uptake; , oxygen uptake.

Fig. 2. Effect of adding crude extract at intervals during the reaction on the oxidation of sulphide, measured as in Fig. I. Reaction mixture as for Fig. i. Additions were as indicated: (i) sulphide at the concentrat ions shown, (2) crude extract containing 5 mg protein. A, fur ther addition of crude extract during Stage i ; B, fur ther addition of crude extract during Stage 2 ; C, oxidation of IO -~ M sulphide.

Addition of further amounts of crude extract during the reaction increases the rate of Stage i (Fig. 2,A) but has no effect on Stage 2 (Fig. 2,B). If the electrode reading of 500 mV in Stage 2 was in fact due to lO -8 M sulphide, then the addition of more crude extract at this point should alter the reading, because the sensitivity of the electrode is sufficient to respond to the oxidation of as little as lO -8 M sulphide (Fig. 2, C). That this does not happen, must mean that no free sulphide is present. The reading in Stage 2 may be accounted for by the formation of an intermediate compound.

Sulphide was effectively oxidised by cell-free extracts over the pH range 5.0-9.0. Boiled preparations of T. concretivorus and extracts of Azotobacter vinelandii did

not utilise sulphide. Since sulphide was enzymically oxidised in less than a minute, loss of sulphide to the atmosphere was negligible.


118 D. J. W. MORIARTY, D. J. D. NICHOLAS

Effect of substrate and enzyme levels The rate of oxidation (Stage I) depends on the relative amounts of sulphide and

extract used (Fig. 3). The Km value determined by a double reciprocal plot is 2. lO -6 M sulphide. There is a linear relation between enzyme concentration and the rate of sulphide oxidation.

The sulphide oxidising activity is associated with the membrane fraction which contained about 90% of the activity. After centrifuging the crude extract at 224 ooo × g for 2 h the enzyme activity was confined to the pellet. The addition of the supernatant fraction to the pellet did not affect the rate of oxidation.

Oxygen requirement Oxygen is necessary for the reaction, since under anaerobic conditions there was

no utilisation of sulphide, as measured by the electrode. A rapid O 8 uptake commenced soon after the start of the Stage i oxidation of sulphide by crude extracts, and decreased to a slower uptake of O~ during Stage 2 (Fig. I).

Electron transfer during sulphide oxidation Cytochromes of the c, b, and a types, reduced immediately on adding sulphide,

were reoxidised by O~ (Fig. 4). They became fully reduced only when 02 was depleted, or excess sulphide added because of an active cytochrome oxidase in the membrane fraction.

A bsorption spectra An increase in absorbance over the range 28o-4oo m/~ occurred when sulphide

was added to whole cells or to crude extracts. When small quantities of sulphide were added, maximum absorption occurred at about 31o m/~, but with further additions, this shifted to around 330 m/~ (Fig. 5). This absorption band will be referred to subsequently as the 300-400 m~ band.

On adding a saturated solution of iodine in potassium iodide to the crude extract,

3C

2(

"6

o -o o

lb 2'o 3b Sulphide (pM]

430 415 1442

/ 55o ~22 ~ ~s8 697

,j

400 4;0 5C)0 550 660 650 ~,(mlJ)

Fig. 3. Effect of var ious a m o u n t s of su lphide and crude ex t rac t on t he ra te of ox ida t ion of su lphide as measu red by the su lphide electrode (see METHODS AND MATERIALS). Reac t ion m i x t u r e as for Fig. I. O - - O , io m g prote in in crude ex t rac t ; & - - • , 5 m g protein.

Fig. 4. Aerobic difference s p e c t r u m of m e m b r a n e fract ion (2 m g protein/ml) oxidised versus reduced wi th 20/zmo!es sulphide.

Biochim. Biophys. Acta, 184 (1969) I I 4 - I 2 3


it became turbid and the band broadened through the visible region of the spectrum with a peak around 350 m~. These are characteristics of elemental sulphur.

The intensity of the 300-400 m~ band is related to sulphide concentration and not to the amount of crude extract present. Large absorption maxima were obtained by successive additions of small quantities of sulphide (about 2 ~moles/mg protein) to whole cells, crude extracts or to membrane fractions. The i 44ooo×g supernatant fraction, freed of membrane fragments by centrifuging at 224 ooo ×g for 2 h did not form the 3oo--4oo-m ff band.

330

]O.lA / . \ Io.lA f ~

( -

2gO ~ o 3go ado 460 25'o 30o 25o 40o 450

Fig. 5. Difference spectra of crude extract (2 mg protein/m1) oxidised versus reduced with sulphide. , 5 / ,moles $2-; - - , i o / ,mo le s S 2-. Note: The pronounced peaks in the ultraviolet

are effects similar to 'solvent cut-off', and absorbances at shorter wavelengths than the max ima of these peaks have no real meaning (see DISCUSSION).

Fig. 6. Effect of added cytochrome c on the difference spectra of membrane fraction (2 mg protein/ml). Oxidised versus reduced with IO /*moles sulphide. , membrane fraction only; . . . . . . , membrane fraction with native cytochrome c; - - , membrane fraction with m a m m a - lian cytochrome c. Note: The pronounced peaks in the ultraviolet are effects similar to 'solvent cut-off', and absorbances at shorter wavelengths than the max ima of these peaks have no real meaning (see DlSCUSSlOX).

When sulphide was added to membrane fractions depleted of cytochrome c by repeated t reatment in a French Press followed by centrifuging and washing, the 300- 4oo-mtz band was small (Fig. 6, Curve A). On recombining these membrane fractions with the purified bacterial cytochrome c, the 3oo-4oo-mff band absorption developed normally (Fig. 6, Curve B) and the cytochrome c was rapidly reduced. When mammalian cytochrome c, however, was substituted for the native component in this experiment, it was immediately reduced on adding sulphide, but the absorption band formed had a maximum at 305 m/~ (Fig. 6, Curve C). This absorbance did not change on adding further amounts of sulphide. The chemical reduction of mammalian cytochrome c by sulphide was slow, and no increase in absorption occurred at 305 mff. The purified native cytochrome c, however, was readily reduced chemically by sulphide, but there was no increase in absorption around 320 mff.

The rate of increase in absorbance at 320 m~ was dependent on the concentration of sulphide. The Km was 2. lO -6 M sulphide. These results are similar to those obtained by the electrode method. There was a lag period between the addition of sulphide and the appearance of an absorption band around 320 mff (Fig. 7). At about I /~mole S2-/mg protein, this period was about 5 sec, although all sulphide had been consumed in this time. At higher sulphide concentrations the lag period increased. The half-time for full development of the 3oo-4oo-m ff band was considerably greater than that for sulphide consumption. Thus the 3oo--4oo-m/~ band is not due to the first product of Stage I oxidation. Extracts of T. thioparus and T. thiooxidans also oxidise sulphide with the formation of the 3oo-4oo-m ~ band.

Biochim. Biophys. Acta, 184 (I969) I I 4 - I 2 3

120 D. j . w . MORIARTY, D. J. D. NICHOLAS

Effect of inhibitors Table I summarises the effect of inhibitors on sulphide oxidation. The inhibition

by sodium diethyldithiocarbamate and by Tris-HC1 was not reversed by dialysis against phosphate buffer. There was little reduction of the cytochromes after adding these compounds, indicating an inhibition of electron flow before the cytochrome sequence.

CO and sodium azide did not restrict 0 2 uptake during Stage I and had no effect on the spectra of cytochrome b or a, indicating that the cytochrome oxidase was not affected. The slow 02 uptake at Stage 2 was, however, partly inhibited by CO, and this effect was reversed by light.

Both cytochrome c of the crude extract and the i2o-fold purified component when reduced were affected by CO, thus the Soret peak increased in intensity at 414 raft, but the 550- and 522-mff bands were depressed (Fig. 8). On adding sulphide to a crude

T A B L E I

INI-IIBITORS OF S U L P H I D E O X I D A T I O N

3 ml reac t ion m i x t u r e s conta ined crude e x t r a c t (2 mg protein), 5 o mM phospha te buffer (pH 7.o) and 200 ffM EDTA, and I /tM Na2S. The crude e x t r a c t was incuba ted for 15 rain pr ior to react ion wi th compounds (1)-(3) a t 5 raM. CO was flushed th rough crude e x t r a c t for 5 mil l before add ing I ffM S 2-.

I n h i b i t o r s % I n h i b i t i o n

S u l p h i d e O ~ A bsorbance c o n s u m p t i o n up tahe increase Stage z Stage I at 320 m,u

I. Sodium d i e t h y l d i t h i o c a r b a m a t e 85 85 90 2. Tris-HC1 65 65 9 ° 3. Sod ium azide o o 8o 4. CO o o 80 5. C O + t u n g s t e n l igh t for 2 min o o o

414

.1 A ~ •

Q 5 10 15 20 25 30 Tirne [see}

. . t,~ 522 550

3gO 460 4~o 56o 5.~o ;k(rn}J) Fig. 7. A t ime course for su lphide ox ida t ion and appearance of the 3oo-4oo-m/~ band. Reac t ion m i x t u r e in each case: 5 ° mM phospha te buffer (pH 7.o), 200/~IV[ Na-EDTA, io ffM S 2- and crude e x t r a c t (5 mg protein) . I n c u b a t i o n s were a t 25 °. , su lphide concen t ra t ion ; , absorbance a t 320 raft. Note : The absorbance a t 320 m/~ reaches a cons t an t va lue af ter abou t 90 sec.

Fig. 8. Effect of CO on the absolu te spec t ra of cy tochrome c purif ied from T. concret ivorus. • . . . . . , oxidised ; , reduced ; - - , + CO.

B i o c h i m . B i o p h y s . Ac ta , 184 (1969) 114-123


extract flushed first with CO, the 3oo-4oo-mt~ band peak did not develop, but on ex- posing to a bright tungsten light for 2 rain, the cytochrome c reverted to its reduced form, and the 3oo-4oo-mt, band developed normally. Thus CO combines with native cytochrome c in this preparation preventing the formation of the compounds with the 3oo-4oo-mtz band.

The following inhibitors at 5" lO-8 M were without effect on sulphide oxidation : Na-EDTA, iodoacetate, 2,2'-dipyridyl, o-phenanthroline and 8-hydroxyquinoline.

The membrane fraction contained about 0.3 t~g copper and o.15 t*g iron per mg protein.

Acetone extraction Sulphide was not oxidised by either the acetone-soluble or -insoluble portions

of the membrane fractions as measured by the sulphide electrode. When these two fractions were recombined, however, enzymic oxidation occurred immediately. The acetone-insoluble fraction was inactivated on boiling, but this t reatment had no effect on the acetone-soluble portion. Cytochrome reduction and the development of the 3oo-4oo-mt~ band occurred only when both fractions were recombined.

DISCUSSION

A notable feature of these results is that free sulphide is rapidly utilised by membrane fragments with a concomitant electron transfer via the cytochrome system to 0 2 as a terminal acceptor. This indicates that the first reaction (Stage I) is an oxidative process. The manometric techniques used by previous workersa, 6 to measure O 3 uptake associated with sulphide oxidation, did not distinguish the early enzymic effects observed here by the sulphide and O 3 electrode procedures and by spectropho- tometric methods. The kinetic data for sulphide oxidation further support the control experiments with boiled preparations in showing that, contrary to Adair's work, these oxidative reactions are mediated by enzymes.

I t is of interest that, in the acetone-soluble material extracted from the membrane fraction, there is a heat-stable factor essential for sulphide oxidation. At present, the nature of this compound is unknown.

The 3oo-4oo-mt, band is clearly due to early intermediates of sulphide oxidation, but the results indicate that it is not the first product of Stage I. The shift to longer wavelengths, and the increase in intensity of the bands with time, on adding more sulphide, suggest that more than one sulphur compound is formed. Polysulphides absorb in this region, and their absorption bands not only increase in intensity but also shift to a longer wavelength as the number of conjugated sulphur atoms increases 1°.

Caution is needed in interpreting the 3oo-4oo-mt, band absorption spectra. The intense protein and nucleotide absorption at wavelengths shorter than 3o0 m/z reduces the energy reaching the photomultiplier to such an extent that an effect similar to the phenomenon of 'solvent cut-off' occurs. The decrease in absorbance at wavelengths shorter than the ultraviolet maxima (see Figs. 5 and 6) is due to this effect, and thus the spectra below this point have no real meaning. In other words, the compounds causing the increase in absorption between 300 and 400 mr* may not themselves, in the pure state, have absorption maxima in this region. I t is not possible to dilute the crude extract to the point where protein absorption does not interfere with the spec-


122 D. J. W. MORIARTY, D. J. D. NICHOLAS

t rum because the enzyme concentration becomes too low for significant oxidation of sulphide. Similarly, dilution of the reference and sample cell contents after the formation of the 30o-4o0 my band to show a change in absorption below 300 m/~ is not possible with the spectrophotometer because the absorbance of the 3oo-4oo-m/, band is very much less than the protein absorbance. Hence these peaks are characteristic of absorption difference spectra of the products of enzymic sulphide oxidation, and it is convenient to refer to them as such. I t should be noted that 320 m/~, the wavelength chosen for the kinetic studies, is higher than the ultraviolet maximum observed under these assay conditions.

I t is likely then, that the absorbance of the compounds with the 3oo-4oo-mt~ absorption actually increases with decreasing wavelength, which is a characteristic of the polysulphides. The likely formation of elemental sulphur on oxidation with iodine with the concomitant broadening and increase in absorption between 300 and 500 m/z supports this deduction.

The binding of CO to the bacterial cytochrome c and the concomitant inhibition of the formation of the 3oo-4oo-m ~ band by CO indicates a possible binding of sulphur intermediates to this cytochrome. On substituting mammalian cytochrome c for the bacterial one, only a small absorption occurred between 3o0 and 40o mr* which was not affected by CO. The nature of these intermediates will be discussed more fully in a subsequent paper.

Because CO inhibits the development of the 3oo-4oo-m~ absorption but has no effect on the rate of sulphide oxidation (Stage I), another intermediate must be formed before those associated with the 3oo--4oo-m/~ absorption. I t is of interest that in the presence of CO, there is a small absorption between 280 and 350 my.

TRUDINGER 11 has postulated that the initial reaction of sulphide oxidation in- volves the splitting of a disulphide bond to form thiols, but this concept is difficult to reconcile with the data presented here. The rapid decrease in electrode potential to low levels on adding enzyme to the reaction mixture would not occur if thiols were formed because the response of the electrode to these compounds is similar to the response in the presence of inorganic sulphide. Moreover, the addition of ferric chloride or potassium ferricyanide, which readily oxidise thiols, had no effect at this stage.

Any compound which complexes with silver will alter the electrode potential, and so it is possible that the reading at Stage 2 of sulphide oxidation is a composite of many intermediates.

Since the copper-chelating agent, sodium diethyldithiocarbamate inhibits sulphide oxidation and the membrane fraction is enriched in Cu, it is likely that a copper-protein binds the sulphide initially, thus facilitating its reaction with the electron transfer chain. The low Kra value of IO -* M for Stage I is in keeping with the high affinity of copper for sulphide. The problem of whether sulphur polymerises at this stage, or after interaction with cytochrome c, is now being investigated.

This interpretation of sulphide oxidation for T. concretivorus is also applicable to T. thiooxidans and T. thioparus.

NOTE ADDED IN PROOF (Received May ist, 1969)

The heat-stable and acetone-soluble factor necessary for sulphide oxidation has been identified as ubiquinone.



R E F E R E N C E S

I C. D. PARKER AND J. PRISK, J. Gen. Microbiol., 8 (1953) 344- 2 W. VISHNIAC AND M. SANTER, Bacteriol. Rev., 21 (1957) 195. 3 J. LONDON AND S. C. RITTENBERG, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1183. 4 J- LONDON, Science, 14o (1963) 409. 5 M. VAN POUCKE, Antonie van Leeuwenhoek J. Microbiol. Serol., 28 (1962) 235. 6 F. W. ADAIR, J. Bacteriol., 92 (1966) 899. 7 H. R. J. LOVI~LOCK AND D. J. D. NICHOLAS, Arch. Mikrobiol., 61 (1968) 3o2. 8 .'~. S. NAIK AND D. J. D. ~ICHOLAS, Biochim. Biophys. Acta, 118 (1966) 195. 9 R. F. ITZHAKI AND D. M. GILL, Anal. Biochem., 9 (1964) 4 ol.

io J. E. BAER AND M. CARMACK, J. Am. Chem. Soc., 71 (1949) 1215. I I P. A. TRUDIN6ER, Rev. PureAppl . Chem., 17 (1967) i.


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Enzymic sulphide oxidation by Thiobacillus concretivorus