THE INITIAL PRODUCT AND PROPERTIES OF THE SULFUR OXIDIZING

22 BIOCHIMICA ET BIOPHYSICA ACTA BBA 65428 THE INITIAL PRODUCT AND PROPERTIES OF THE SULFUR-OXIDIZING ENZYME OF THIOBACILLI ISAMU SUZUKI AND MARVIN SILVER Department of Microbiology , University of Manitoba, Winnipeg (Canada) (Received December 27th, 1965) SUMMARY I The sulfur-oxidizing enzyme of Thiobacillus thioparus was purified a n d the p r o p e r t i e s were studied Catalase (EC 1.11.1.6) a n d , ' - d i p y r i d y l p r o t e c t e d G S H from o x i d a t i o n to GSSG during incubation The Km for G S H u n d e r these conditions was mM Non-heine iron t i g h t l y b o u n d to the p r o t e i n was identified as an e n z y m e c o m p o n e n t b y the inhibition studies with m e t a l chelators a n d direct d e t e r m i n a t i o n s of iron in the purified enzyme p r e p a r a t i o n The initial p r o d u c t of sulfur o x i d a t i o n b y the enzyme of 7" thioparus a n d Thiobacillus thiooxidans was identified as sulfite b y t r a p p i n g with f o r m a l d e h y d e I t is p r o p o s e d t h a t the enzyme is an i r o n - c o n t a i n i n g oxygenase which oxidizes e l e m e n t a l sulfur to sulfite with G S H as cofactor: S + 02 + H20 GSFI> H2SO3 (I) while thiosulfate is formed t h r o u g h a secondary, n o n - e n z y m a t i c r e a c t i o n : H2SO3 + S -> H2S203 (2) A possible role of this e n z y m e in the o x i d a t i o n of sulfur a n d thiosulfate b y thiobacilli is discussed INTRODUCTION The thiobacilli are c h e m o a u t o t r o p h i c b a c t e r i a t h a t derive energy for g r o w t h from t h e o x i d a t i o n of reduced inorganic sulfur compounds A l t h o u g h most thiobacilli are capable of oxidizing b o t h sulfur a t o m s of thiosulfate (S-SO32-) to sulfate, the m e c h a n i s m of o x i d a t i o n of the outer sulfur a t o m (S-) has never been fully explained1, Our s t u d y on the e n z y m a t i c o x i d a t i o n of e l e m e n t a l sulfur b y Thiobacillus thiooxidans led to the isolation of an e n z y m e which oxidized e l e m e n t a l sulfur to thiosulfate w i t h G S H as cofactor: s + 02 + H~O ~ S H H~S203 Biochim Biophys Acta, i22 (1966) 22-33 (31 SULFUR-OXIDIZINGENZYME OF THIOBACILLI 23 The enzyme was tentatively identified as an oxygenase from the results of 180zexperiments In this report we present evidence that Eqn is actually a sum of Eqns I and and sulfite rather than thiosulfate is the initial oxidation product of sulfur as was suggested previously Since the reaction of Eqn was very rapid under experimental conditions where sulfur was present in large excess, it was not possible to detect any free sulfite as such Sulfite was therefore trapped as a formaldehydebisulfite complex and determined after hydrolysis of the complex with alkali Cell-free extracts of Thiobacillus thioparus grown on thiosulfate also catalyzed the reaction of Eqn This organism proved to be a better source of the enzyme than T thiooxidans because of its higher growth rate and the invariably high activity of the enzyme in cell-free extracts The properties of the enzyme purified from T thioparus were therefore studied in an effort to clarify the nature of reaction and to assign a possible role to the enzyme in the oxidation of the outer sulfur atom of thiosulfate MATERIALSAND METHODS Organism T thiooxidans (ATCC 8o85) was grown as described previously T thioparus (ATCC 8158 ) was grown in 15-I carboys with aeration Each carboy contained IO of the Starkey's medium No (ref 5): io g Na~S20 a.5H20, ° g K H z P Q , ° g K~HPO4, 0-7 g CaC12, g (NH4)~SO4, 0.2 g FeCI~.6H20, 0.2 g MnSO4.2H~O, ml phenol red (2%) and distilled water to make up a total volume of io after the adjustment of pH to 7.0-7.5 with lO°/0 K2CO z With a 2-3% inoculum the carboys were incubated at 25 ° for 5-7 days with periodic neutralization with lO°/0 K2CO z to around pH Cells were collected in a Sharples centrifuge, washed and decanted several times with 0.2 M Tris-HC1 (pH 7.4) to remove residual sulfur The washed cells were stored at 20 ° until used Yield was 5-8 g wet weight of cells per carboy Preparation of cell-free extracts The extracts of T thiooxidans were prepared as described T thioparus cells were suspended in 0.2 M Tris-HC1 buffer of pH 7.4 (I5%, w/v) and sonicated in a Io kcyles Raytheon oscillator for 20 under N atmosphere The cell-flee extract was obtained after removal of cell debris by centrifugation at 23 500 × g for 20 Purification of the enzyme The sulfur-oxidizing enzyme of T thiooxidans was purified as described previously ~ except that the final ethanol precipitation of enzyme was carried out at pH 5.0 instead of pH 7.5 The enzyme was collected between the ethanol concentrations of 15% and 30% after overnight incubation at IO ° The enzyme of T thioparus was purified by the same procedures Further purification was achieved by adsorption of the enzyme to D E A E cellulose and subsequent elution with a Tris-HC1 buffer The 30% ethanol precipitate suspended in 0.05 M Tris-HC1 buffer of pH 7.5 (6 mg enzyme protein/ml) was stirred for 30 at ° with an equal volume of DEAE-cellulose in 0.05 M Tris-HC1 buffer (pH 7.5) After centrifugation at 23 500 × g for 20 min, the DEAE-cellulose was Biochim Biophys Acta, 122 (1966) 22-33 24 I SUZUKI, M SILVER washed twice w i t h t h e same volume of buffer at o.o5 M a n d o.I M, respectively The enzyme was finally eluted from the cellulose with 0.25 M Tris-HC1 (pH 7.5) Protein was d e t e r m i n e d b y the m e t h o d of LOWRY et al.* Enzyme assays The o x i d a t i o n of sulfur b y the e n z y m e was followed m a n o m e t r i c a l l y at °° in a W a r b u r g a p p a r a t u s The reaction m i x t u r e contained, unless otherwise indicated, 500 # m o l e s of Tris-HC1 (pH 7.8), 48 mg of sulfur, / , m o l e s of GSH, 250 # g of catalase (EC I I I I ) , 0.2 ~mole of 2,2'-dipyridyl, enzyme (0.6 mg of the o % e t h a n o l precipitate) and w a t e r to m a k e a t o t a l volume of 2.0 ml The reaction was s t a r t e d b y the a d d i t i o n of GSH A f t e r 21o rain thiosulfate was d e t e r m i n e d b y the m e t h o d of SORBO as described a Determination of thiosulfate and sulfite in the initial product experiments Thiosulfate a n d sulfite were d e t e r m i n e d b y two i n d e p e n d e n t procedures The m e t h o d A was a modification of t h a t of GOLDMAN AND YAGODA8 where thiosnlfate was t i t r a t e d with iodine in the presence of f o r m a l d e h y d e , a n d sulfite was then t i t r a t e d with iodine after the dissociation of the f o r m a l d e h y d e bisulfite complex with sodium carbonate, i ml of the r e a c t i o n m i x t u r e was t r e a t e d with I ml of u r a n y l acetate (0.8%) to remove proteins a n d GSSG To the s u p e r n a t a n t after centrifugation o.I ml of % f o r m a l d e h y d e was a d d e d a n d after rain t h e m i x t u r e was t i t r a t e d with O.Ol N iodine to a blue end p o i n t w i t h a s t a r c h indicator ml of s o d i u m c a r b o n a t e buffer (80 g sodium c a r b o n a t e dissolved in 500 ml water, after a d d i t i o n of 20 ml of glacial acetic acid d i l u t e d to I 1) were t h e n a d d e d a n d the solution was t i t r a t e d again to the end p o i n t w i t h iodine The first t i t r a t i o n value gave t h e a m o u n t of thiosulfate and the second the a m o u n t of sulfite In the m e t h o d B thiosulfate was d e t e r m i n e d according to SORBO7 as described p r e v i o u s l y a n d sulfite b v a modification of t h e m e t h o d used b y TRfflPER AND SCHLEGEL m e a s u r i n g the f o r m a t i o n of f u c h s i n - s u l f i t e - f o r m a l d e h y d e complex I n order to avoid interference b y f o r m a l d e h y d e present in reaction m i x t u r e s , samples for analysis were frozen at 2o ° for several hours before the d e t e r m i n a t i o n s in the m e t h o d B This t r e a t m e n t p r e s u m a b l y p o l y m e r i z e d excess f o r m a l d e h y d e , t h u s prev e n t i n g its interfering with the d e t e r m i n a t i o n s F o r sulfite d e t e r m i n a t i o n a 3-ml sample c o n t a i n i n g o.I to 0.3 # m o l e of sulfite was t r e a t e d with I ml of I °/o zinc a c e t a t e a n d was centrifuged To the s u p e r n a t a n t o.I ml of I M N a O H was a d d e d a n d the m i x t u r e was allowed to s t a n d for 0.5 h to dissociate t h e f o r m a l d e h y d e - b i s u l f i t e complex The m i x t u r e was then m a d e u p to ml w i t h w a t e r a n d 0.5 ml of a fuchsin solution (4 ° mg fuehsin in IOO ml of 12.5% H2SO4) was added, followed, after IO rain, b y 0.05 ml of % f o r m a l d e h y d e A f t e r a n o t h e r 20 the a b s o r p t i o n was m e a s u r e d at 570 m # in a U n i e a m SP 700 s p e c t r o p h o t o m e t e r S t a n d a r d d e t e r m i n a t i o n s with known a m o u n t s of sulfite were always carried out simultaneously Determination of iron, copper and labile sulfide Non-heine iron in the enzyme p r e p a r a t i o n was d e t e r m i n e d b y m e a s u r i n g t h e a b s o r p t i o n of the ferrous , ' - d i p y r i d y l complex b y a modification of t h e m e t h o d s by RAJAGOPALAN AND HANDLER 1° a n d MASSEY 11 A o.5-ml sample was t r e a t e d with 0.05 ml of % triehloroaeetic acid to release the iron from the enzyme To the Biochim Biophys Acta, 122 (1966) 22-33 SULFUR-OXIDIZING ENZYME OF THIOBACILI,I 25 supernatant after centrifugation were added 1.5 ml of water and o.2 ml of saturated ammonium acetate After o.5-h incubation with o.i ml of o.oi M 2,2'-dipyridyl, the absorbance was measured at 520 m# in a Unicam SP 700 spectrophotometer Copper was determined in a Perkin-Elmer Model 303 Atomic Absorption Spectrophotometer Absorption by the enzyme preparation was measured at 325 m# and compared to standards of i, 3, and parts per million copper Labile sulfide was determined by a modification of the method of FOGO AND POPOWSKY12 A o.65-ml sample was treated with an equal volume of 2% zinc acetate and centrifuged To the supernatant, 2.5 ml of o i °/o p-aminodimethylaniline sulfate in 5.5 M HC1 and ml of 0.023 M FeCI~ in 1.2 M HC1 were added in a screw cap test tube and shaken After 3o min, the intensity of methylene blue formed was measured at 670 m# in a Unicam SP 700 spectrophotometer Chemicals All the chemicals used were obtained from commercial sources Catalase (liver, times crystallized), GSH, FAD and FMN, Sigma Chemical Co ; sodium diethyldithiocarbamate, Fisher Scientific Co ; 2,2'-dipyridyl, British Drug Houses Ltd ; Cleland's reagent (dithiothreitol), California Corporation for Biochemical Research; atebrin (quinacrine dihydrochloride), Mann Research Laboratories, Inc ; precipitated sulfur powder, Baker Chemical Co All the reagents including buffers were prepared in glass-distilled water The elemental sulfur suspension used as substrate for oxidation was prepared by sonication for h of a sulfur suspension in water containing 0.05% Tween-8o After sonication the sulfur suspension was extensively dialysed to remove any contaminating metal ions Metals used for the inhibition studies were: Fe(NH4)2(SO4) 2, FeClu, CuSQ, CoC12, ZnSO 4, MgSO and MnSO TABLE I PURIFICATION OF THE SULFUR-OXIDIZING ENZYME FROM T thioparvts The e n z y m e a c t i v i t y was d e t e r m i n e d u n d e r s t a n d a r d c o n d i t i o n s The a m o u n t of p r o t e i n us e d was as follows: c r u d e cell-free e x t r a c t , 15 m g ; p H 5.0 s u p e r n a t a n t , i i m g ; 3o~o e t h a n o l pre c i pi t a t e , 0.6 m g ; or D E A E - c e l l u l o s e - t r e a t e d e n z y m e , e l u t e d w i t h o.25 M Tris-HC1 (pH 7.5), 0.4 mg Fraction Total protein (rag) Specific activity ~ Cell-free e x t r a c t p H 5.0 s u p e r n a t a n t 3O~o e t h a n o l p r e c i p i t a t e DEAE-cellulose fraction 2650 188 15o ioo 0.43 16.6 34.2 53-5 * Specfic a c t i v i t y was e x p r e s s e d as t h e n u m b e r o f / * m o l e s of t h i o s u l f a t e f o r m e d in 21o m i n p e r m g of pro tein RESULTS Enzyme purification and stability Results of purification of the sulfur-oxidizing enzyme from T thioparus are given in Table I Biochim Biophys Acta, 122 (1966) 22-33 26 I SUZUKI, M SILVER Crude extracts of T thioparus had relatively little activity, probably due to some inhibitory substances which were largely removed in the first purification step The 30% ethanol precipitate fraction, which was used in all the experiments unless otherwise indicated, was purified approx 24-fold compared to the cell flee extract This calculation was made on the assumption that the total activity of the cell-flee extract was equal to that of the pH 5.0 supernatant This degree of purifification was consistent All fractions could be stored at 20 ° at pH 7.5-8.0 for at least weeks without loss of activity with the exception of the DEAE-cellulose fraction, which lost activity when frozen and thawed, and was almost completely inactivated when stored at 20 ° for longer than days No fraction was stable to freezing at pH 5.0 Both cell-flee extracts and the 15-3o% ethanol precipitate fraction lost approximately half the activity when stored at ° overnight Half the protein could be removed by heating the 15 30% ethanol precipitate fraction at 5°0 for This treatment, however, did not raise the specific activity Little protection was afforded by the addition of sulfur and GSH, thiosulfate, or sulfite The activity was almost totally destroyed by heating the enzyme at 60 ° for Attempts to purify the enzyme with ammonium sulfate precipitation were unsuccessful because of severe loss of activity Linear gradient chromatography of the 15 30% ethanol precipitate through a DEAE-cellulose column with Tris-HC1 (pH 7-5, 0.05-0-2 M) resulted also in considerable loss of activity Sulfite, GSH, thiosulfate, 2-mercaptoethanol, gelatin, or Cleland's reagent, gave little protection in this treatment Effect of 2,2'-dipyridyl and iron Since many oxygenases contain non-heine iron as cofactor, the effect of 2,2'dipyridyl on sulfur oxidation by the enzyme was studied As shown in Fig i, 2,2'dipyridyl at low concentrations stimulated the sulfur oxidation, especially after prolonged incubation periods Without the chelator the reaction slowed down considerably after I h The fact that this phenomenon was not due to the inactivation of enzyme was demonstrated by a later, secondary addition of GSH which restored the rapid initial rate of oxidation Thus it is apparent that 2,2'-dipyridyl was protecting GSH from destruction during incubations Since the enzyme preparation (30 % ethanol precipitate) contained iron as observed by the development of a red irondipyridyl complex in the reaction mixtures, the destruction of GSH was probably due to a known oxidation reaction to GSSG catalyzed by iron and oxygen 13 In fact Fe 2+ and Fe 3+ ions at lO -4 M were strongly inhibitory, the inhibition occurring only after prolonged incubation periods Inhibition of the initial oxidation rate at higher concentrations of 2,2'-dipyridyl was apparently due to the removal of iron from the enzyme The addition of GSH did not restore the full activity of enzyme Since the 2,2'-dipyridyl concentration of lO -4 M was not inhibitory, this amount of dipyridyl was added to the reaction mixture routinely Effect of other chelating agents and metal ions As shown in Fig 2, o-phenanthroline had a very similar effect as 2,2'-dipyridyl EDTA and diethyldithiocarbamate were inhibitory The inhibition by EDTA was Biochim Biophys Acta, 122 (1966) 22-33 S U L F U R - O X I D I Z I N G E N Z Y M E OF T H I O B A C I L L I 27 S z 0~,~ MOLES 35 ~,~ 34.8 30 32.4 25 214 J ~ zo i 60 120 i 180 t / 240 300 ~IME OF tNCUSATION (MINUTES) F i g I E f f e c t o f , ' - d i p y r i d y l , ' - D i p y r i d y l w a s p r e s e n t a t t h e c o n c e n t r a t i o n 21o m i n G S H ( / ~ m o l e s ) w a s a d d e d a g a i n t o e a c h r e a c t i o n m i x t u r e indicated At p r o b a b l y due to t h e s t i m u l a t o r y action on the o x i d a t i o n of sulfidel4, z5 a n d was p o s s i b l y caused b y t h e r e m o v a l of G S H from t h e s y s t e m at the level of g l u t a t h i o n e polysulfide E D T A d i d n o t cause the i n a c t i v a t i o n of enzyme, since a s e c o n d a r y a d d i t i o n of G S H r e s t o r e d the initial r a p i d r a t e of oxidation The effect of d i e t h y l d i t h i o c a r b a m a t e r e m a i n s u n e x p l a i n e d The i n h i b i t i o n was n o t reversed b y the a d d i t i o n of Cu 2+ ions Zn 2+, Co 2+ a n d Cu 2+ ions at 10 -4 M were found i n h i b i t o r y e i t h e r in the presence or absence of 10 -4 M , ' - d i p y r i d y l The degree of i n h i b i t i o n v a r i e d a m o n g S20~MOLES 3C O- Pt4I~IANI~ROUtE 2:3.7 :2¢-O~PYRIOYL " 23.7 2~ NO CHELATINGAGENT 20.0 ~=_OTA SODIUMOtETHYLITHIO CARBAMATE !,o 14.4 12.8 o ; 60 ] IZ'O 180 2110 TIME OF INCUBATION (MINUTES) F i g E f f e c t o f v a r i o u s c h e l a t i n g a g e n t s All t h e m e t a l - c h e l a t i n g a g e n t s w e r e p r e s e n t a t lO -4 M Biochim Biophys Acta, 122 (1966) 22-33 28 I SUZUKI, M SILVER 3C 2" 240 300 TiME O F I N C U B A T f O N (MIN) Fig R e v e r s a l of H202 i n h i b i t i o n by c a t a l a s e a n d GSH All t h e flasks i n i t i a l l y c o n t a i n e d t h e s t a n d a r d r e a c t i o n m i x t u r e unless o t h e r w i s e i n d i c a t e d W h e n i n d i c a t e d , i o / m o l e s of H202, 25 ° / ~ g of c a t a l a s e or 5/*moles of G S H were added F l a s k i : a d d i t i o n a l G S H a d d e d a t 12o F l a s k 2: no a d d i t i o n s F l a s k 3: c a t a l a s e i n i t i a l l y a b s e n t ; H v c a t a l a s e , a n d a d d i t i o n a l G S H a d d e d a t 6o, 9o, a n d 12o min, r e s p e c t i v e l y F l a s k 4: H202, b u t n o t c a t a l a s e , i n i t i a l l y p r e s e n t ; c a t a l a s e a n d a d d i t i o n a l GSH a d d e d a t 9o a n d 12o min, r e s p e c t i v e l y 02 u p t a k e v a l u e s were cor rected for t h e a m o u n t of o x y g e n e v o l v e d from H~O different enzyme preparations The inhibition was not restored by the addition of GSH and seems, therefore, to be due to inactivation of the enzyme These metals may compete with iron on the enzyme and thus cause inhibition Such a competition was reported on the Zn e~ inhibition ofp-hydroxypyruvate hydroxylase (EC 1.99.1.14) (ref 16) Mg 2+ and Mn 2+ were not inhibitory Effect of catalase and H202 The effects of catalase and H202 on the sulfur-oxidizing enzyme of T thioparus were similar to those found with the T thiooxidans enzyme Catalase stimulated the sulfur oxidation after prolonged incubation periods; H~02 was strongly inhibitory In order to elucidate whether the inhibition by H20 ~ was due to the destruction of GSH or destruction of the enzyme, eatalase, H202 and GSH were added at various times during the reaction As shown in Fig the inhibition by H202 was reversed after subsequent addition of catalase and GSH From these results it is clear that H 2 destroyed GSH, but not the enzyme Catalase, therefore, probably protects GSH from conversion to GSSG by H202 produced non-enzymically during the oxidation of GSH 17 Effect of GSH concentration In the presence of catalase and 2,2'-dipyridyl it became possible to protect GSH effectively during long incubation periods Under these conditions the reaction Biochim Biophys Acta, 122 (1966) 22-33 SULFUR-OXIDIZING ENZYME OF THIOBACILLI S 2)~ES] 40[ Lu 29 / / ,,o~.~" / ,4 >,oI ~ 16.0 / /" _.~.~ ~J'J I / I 5f / j ~ / / - ;.~ ' o i 6o ! ''°/ os - ,20 ~ s,85 ,~o | ~io TIME OF INCUBATION (MINUTES) Fig Effect of GSH concentration proceeded linearly for a considerable length of time with a clear dependency on GSH concentrations (Fig 4) In previous experiments the non-enzymatic oxidation of GSH complicated the results The effect of GSH concentration on the sulfur oxidation gave a simple, linear LINEWEAVER-BURK plot 18 (Fig 5) and the Km for GSH was calculated as 6.3 mM 02 30.2 id o -o.~ ,/ oo oi~ o; I/EGSH'] , ,.~ ,; ~io {rnM) - I Fig LINIWEAVER BuRI~ p l o t o f t h e effect o f G S H c o n c e n t r a t i o n Biochim Biophys Acta, 122 (1966) 22-33 3° I S U Z U K I , M S I L V E R The G S H r e q u i r e m e n t was specific as was the case with the T thiooxidans enzyme Cysteine, - m e r c a p t o e t h a n o l , , - d i m e r c a p t o p r o p a n o l (BAL), sulfide, GSSG a n d ascorbic acid d i d not replace G S H in the reaction Identification of iron as cofactor An effort to p r e p a r e an iron-free e n z y m e p r e p a r a t i o n b y passing a m i x t u r e of enzyme and , ' - d i p y r i d y l (0.I M) t h r o u g h a small column of S e p h a d e x G-25 always resulted in a p r e p a r a t i o n which showed v e r y little a c t i v i t y a n d was only slightly s t i m u l a t e d w i t h 10 or I0 -~ M Fe 2+ ions Reisolation of the enzyme from the m i x ture with a batchwise D E A E - c e l l u l o s e t r e a t m e n t as described in MATERIALS AND METHODS led to a p r e p a r a t i o n which r e t a i n e d over % of the original a c t i v i t y a n d was s t i m u l a t e d slightly b y Fe 2+ ions at IO M as shown in Table I I F e 2+ ions at lO -4 M were i n h i b i t o r y A l t h o u g h the s t i m u l a t i o n was not v e r y m a r k e d , it was c o n s i s t e n t l y observed I n order to eliminate the p o s s i b i l i t y t h a t copper was a cofactor, a similar t r e a t m e n t of the enzyme d i e t h y l d i t h i o c a r b a m a t e (o.I M) m i x t u r e with D E A E cellulose was carried out The a c t i v i t y of the enzyme t h u s t r e a t e d was not s t i m u l a t e d b y Cu e+ ions at lO -5 or IO M a n d was s t r o n g l y i n h i b i t e d b y F e 2+ ions at IO -~ or IO - M F i n a l l y , the content of iron in the purified e n z y m e p r e p a r a t i o n ( D E A E fraction) was d e t e r m i n e d E a c h m g of e n z y m e p r o t e i n c o n t a i n e d 0.087 # m o l e of iron of which 0.055 # m o l e was in the r e d u c e d ferrous state The enzyme also c o n t a i n e d o o i / , m o l e of labile sulfide per mg protein The % e t h a n o l p r e c i p i t a t e fraction h a d 0.048 # m o l e t o t a l iron a n d 0.039 # m o l e ferrous iron per m g protein The labile sulfide c o n t e n t was o.o16 # m o l e per mg The copper c o n t e n t of the enzyme was determ i n e d to be less t h a n 0.002/~mole per rag The s p e c t r u m of the D E A E fraction showed one a b s o r p t i o n p e a k only at 275 m/~ in the u l t r a v i o l e t range a n d no a b s o r p tion in the visible range F M N a n d F A D were i n h i b i t o r y to the o x i d a t i o n of sulfur in a g r e e m e n t with the p r o p e r t y of the T thiooxidans e n z y m e A t e b r i n h a d v e r y little i n h i b i t o r y action F r o m these o b s e r v a t i o n s it is u n l i k e l y t h a t t h e e n z y m e cont a i n s a n y flavin nucleotides or heine c o m p o u n d s as cofaetor TABLE EFFECT II OF IRON ON T H E PARTIALLY IRON-FREE ENZYME T h e r e a c t i o n w a s c a r r i e d o u t u n d e r s t a n d a r d c o n d i t i o n s f o r e n z y m e a s s a y s , e x c e p t t h a t 2,2"dipyridyl was omitted and ferrous ammonium sulfate was added to the concentration indicated E a c h f l a s k c o n t a i n e d o m g o f t h e e n z y m e t r e a t e d as f o l l o w s : t h e ° ~o e t h a n o l p r e c i p i t a t e f r a c t i o n (2.o ml) w a s t r e a t e d w i t h , " - d i p y r i d y l a t a final c o n c e n t r a t i o n o f o i M f o r o.5 h a t °, a f t e r w h i c h i t w a s a d s o r b e d o n D E A E - c e l l u l o s e a n d e l u t e d a s d e s c r i b e d in MATERIALS AND METHODS T h e D E A E - c e l l u l o s e w a s w a s h e d w i t h 4o m l e a c h o f o.o5 M a n d o i M T r i s - H C ( p H 7.5) b e f o r e t h e e l u t i o n o f e n z y m e w i t h t h e s a m e b u f f e r a t o.25 M Final concentration of Fe 2+ 02 uptake S20a 2(l~moles) formation (#moles) Nil IO-8 M lO -4 M 13.o 14 lO 12 14.1 lO Biochim Biophys 4cta, 122 (1966) 2 - 3 31 SULFUR-OXIDIZING ENZYME OF THIOBACILLI TABLE III EFFECT OF FORMALDEHYDE ON PRODUCTS OF THE SULFUR-OXIDIZING ENZYME T h e r e a c t i o n m i x t u r e s c o n t a i n e d in a t o t a l v o l u m e of m l : 500/*moles Tri s -H C (pH 7.8), 25 ° # g c a t a l a s e , / z m o l e s , " - d i p y r i d y l ; 3.0 m g of T thioparus e n z y m e or m g of T thiooxidans e n z y m e (30% e t h a n o l p r e c i p i t a t e ) , 48 m g sulfur, # m o l e s GSH, a n d o t h e r a d d i t i o n s as i n d i c a t e d The i n c u b a t i o n was c a r r i e d o u t a t °0 for 21o m i n in air A, B: T h i o s u l f a t e a n d sulfite were d e t e r m i n e d b y t h e m e t h o d A or B, r e s p e c t i v e l y , as d e s c r i b e d in MATERIALS AND METHODS System O~ uptake (t~moles) S~Oa2(#moles) A B SOa2(#moles) A B 29.1 26.5 25.6 22.4 13 o.o o.o 2.5 28.9 2o.o 13.3 8.0 1.6 o.o o.o 19.4 29.6 21.8 15.9 o.o o.o 19.2 0.5 5-5 lO.5 14 13.5 o.o o.o 0.5 0.5 12-3 16 13.6 o.o o.o 0.5 2.0 2.0 19.6 o.8 18.6 o.6 o.o 2.6 o.o 11.2 11.4 lO 12 8.8 6.6 12 7.0 5.3 0.2 2.6 5.2 2.2 4.6 T thioparus enzyme S + GSH S + G S H + H C H O (IO #moles) S + G S H + H C H O (5 ° #moles) S + G S H + H C H O ( i o o #moles) S + G S H + H C H O (2o0 #moles) S S + H C H O (5 ° #mo les) Na~S203 (20 #moles) + G S H NaS208 (20 # mo les) + G S H + H C H O (5 ° #mo les) GSH T thiooxidans enzyme S + GSH S + G S H + H C H O (5o #moles) S + G S H + H C H O ( i o o #moles) Initial product of sulfur oxidation As shown in Table I I I formaldehyde trapped sulfite during the oxidation of sulfur b y both T thioparus and T thiooxidans enzymes The trapping of sulfite became more efficient with higher concentrations of formaldehyde The enzyme activity was not inhibited appreciably by formaldehyde below 0.05 M as manifested by constant oxygen uptakes In all experiments the amount of oxygen consumed equalled the sum of thiosulfate and sulfite produced in accordance with the Eqns I and TABLE IV NON-ENZYMIC REACTIONS OF SULFUR COMPOUNDS The c o n d i t i o n s were t h e s a m e as in T a b l e I I I , e x c e p t t h a t t h e e n z y m e w a s o m i t t e d System 03 uptake (#moles) $2032(#moles) A B S032(#moles) A B S + GSH S + G S H + H C H O (5o #moles) Na2SaO (20 #mo les) + G S H Na~S20 s (20 #mo les) + G S H + H C H O (5 ° #mo les) S + Na2SO (Io,umoles) S + Na,SO (io#moles) + GSH 5 1.8 0.8 2o.0 i.i 0.8 19.4 o.o i o 0.5 o.o i.i 0.5 20.0 io.o lO.4 19.8 9.6 9.6 1.5 o.o o.o 1.4 o.o o.o Biochim Biophys Acta, 122 (1966) 22-33 32 I S U Z U K I , M S I L V E R Sulfite trapped as the formaldehyde-bisulflte complex could not have come from a secondary decomposition of thiosulfate either chemically or enzymatically through the action of thiosulfate reductase 19, since the amount of sulfite formed from thiosulfate under experimental conditions was quantitatively much smaller (Tables I I I and IV) A small amount of sulfite formed during the incubation of thiosulfate and GSH was probably due partly to a known non-enzymic reaction between the two compounds 19 and partly to thiosulfate reductase activity present in the sulfuroxidizing enzyme preparation as well as in the crude extracts as reported by PECK As shown in Table IV sulfite was completely converted to thiosulfate when incubated with sulfur (Eqn 2) DISCUSSION From the results presented in this and previous papers3, it is concluded that the sulfur-oxidizing enzyme of thiobacilli is an oxygenase containing non-heine iron and possibly also labile sulfide Iron is apparently very tightly bound to the protein since it is only partly removed at 0.I M 2,2'-dipyridyl The removal of iron largely destroys the enzyme activity which is restored only slightly by the addition of ferrous ions at a very low concentration (10 -5 M) Higher concentrations of iron were inhibitory Similar properties were reported for oxygenases with non-heine iron, such as 3,4-dihydroxyphenylacet ate 2,3-0xygenase 2°, pyrocatechase (EC I 13.I I) (ref 2I), steroid Ilfl-hydroxylase 22 and camphor ketolactonase 23 The tight binding of iron to the protein is shown also by the observation that the DEAE-cellulosetreated enzyme releases the iron only after denaturation of the protein with trichloroacetic acid Treatment of the enzyme with 2,2'-dipyridyl without denaturation did not form the colored complex of ferrous ion and dipyridyl It is difficult to estimate the number of iron atoms or labile sulfide atoms bound to each enzyme molecule at present, since our best enzyme preparation is only partially pure It is interesting that the steroid II/~-hydroxylase complex contains, as a component, a non-heine iron protein with labile sulfide 22 From the effects of formaldehyde on the products of sulfur oxidation, it is concluded that the initial oxidation product of sulfur by the sulfur-oxidizing enzyme of thiobacilli is sulfite (Eqn I) and that the thiosulfate formation is due to a secondary, non-enzymic, reaction of sulfite with sulfur (Eqn 2) It is possible that the sulfite formed is not released from the enzyme as free sulfite, but directly converted to thiosulfate by sulfur or to a formaldehyde-bisulfite complex by formaldehyde The implication of the discovery that sulfite rather than thiosulfate is the initial product of sulfur oxidation is that the oxidation of sulfur to sulfate b y thiobacilli m a y be explained by the combination of a sulfur-oxidizing system and a sulfite-oxidizing system, either through adenosine phosphosulfatel, or directly through a cytochrome system 24 The presence of an active sulfur-oxidizing enzyme in T thioparus m a y signify a possible role of the enzyme in the oxidation of outer sulfur atom of thiosulfate molecule observed with whole cells 2,25 I f it is assumed that the initial scission of thiosulfate by GSH leads to sulfur and sulfite instead of sulfide and sulfite as in the original PECK's scheme~, 2, the following reactions m a y be formulated: Biochim Biophys Acta, 122 (1966) 22 33 SULFUR-OXIDIZING ENZYME OF THIOBACILLI SSO32- + G S - - + GSS- + SO32GSS- + 02 + HzO -> G S - + SO 2- + H + 2SO32- + H - - - > S O ~- + e - + H + e - + O3 + H+ > H i O SSOs 2- + 02 + H ~ O - + SO~2- + H + 33 (4) (5) (6) (7) (8) The role of GSH is catalytic in this system whereas it is required in substrate quantities in the mechanism proposed by PECK1,2 The sulfur-oxidizing enzyme catalyses the reaction of Eqn oxidizing glutathione polysulfide If sulfite, not thiosulfate, is the primary product of this oxidation, the oxidation of both sulfur atoms of thiosulfate to sulfate (Eqn 8) may be achieved by a combined action of the thiosulfatecleaving system (Eqn 4), the sulfur-oxidizing enzyme (Eqn 5) and the sulfite-oxidizing system (Eqns and 7) The validity of this hypothesis is currently under investigation In this connection, it is interesting that LONDON AND RITTENBERG26 had to subject the cell-free extracts of a strain of T thioparus to extensive dialysis and Norit A treatment before the achievement of complete oxidation of reduced sulfur compounds to sulfate It is possible that such treatments removed from the extracts heavy metal ions or flavin nucleotides which are inhibitors of the sulfuroxidizing enzyme ACKNOWLEDGEMENTS The authors thank the National Research Council of Canada for a grant in support of the work and Professor H LEES for his interest and encouragement We also acknowledge the help of Dr K RAMLAL,Department of Geology, University of 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