THE INITIAL PRODUCT AND PROPERTIES OF THE SULFUR OXIDIZING

I and 2 and sulfite rather than thiosulfate is the initial oxidation product of sulfur as was suggested previously 3.. thiooxidans because of its higher growth rate and the invariably hi

BBA 65428

T H E I N I T I A L P R O D U C T A N D P R O P E R T I E S O F T H E S U L F U R - O X I D I Z I N G

E N Z Y M E O F T H I O B A C I L L I

ISAMU SUZUKI AND MARVIN SILVER

Department of Microbiology , University of Manitoba, Winnipeg (Canada)

(Received December 27th, 1965)

SUMMARY

I The s u l f u r - o x i d i z i n g e n z y m e of Thiobacillus thioparus was purified a n d the

p r o p e r t i e s were s t u d i e d

2 Catalase (EC 1.11.1.6) a n d 2 , 2 ' - 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

G S S G d u r i n g i n c u b a t i o n The Km for G S H u n d e r these c o n d i t i o n s was 6 3 mM

3 N o n - h e i n e iron t i g h t l y b o u n d to t h e 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 t h e i n h i b i t i o n s t u d i e s w i t h m e t a l c h e l a t o r s a n d d i r e c t d e t e r m i n a t i o n s

o f iron in t h e purified e n z y m e p r e p a r a t i o n

4 The i n i t i a l p r o d u c t of sulfur o x i d a t i o n b y the e n z y m e of 7" thioparus a n d

Thiobacillus thiooxidans was i d e n t i f i e d as sulfite b y t r a p p i n g w i t h f o r m a l d e h y d e

5 I t is p r o p o s e d t h a t t h e e n z y m e is a n i r o n - c o n t a i n i n g o x y g e n a s e w h i c h oxi- dizes e l e m e n t a l sulfur to sulfite w i t h G S H as c o f a c t o r :

while t h i o s u l f a t e is f o r m e d t h r o u g h a s e c o n d a r y , n o n - e n z y m a t i c r e a c t i o n :

6 A possible role of t h i s e n z y m e in t h e o x i d a t i o n of sulfur a n d t h i o s u l f a t e b y

t h i o b a c i l l i is discussed

INTRODUCTION

The t h i o b a c i l l i 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 d e r i v e e n e r g y for g r o w t h

f r o m t h e o x i d a t i o n of r e d u c e d i n o r g a n i c sulfur c o m p o u n d s A l t h o u g h m o s t t h i o -

b a c i l l i are c a p a b l e of o x i d i z i n g b o t h sulfur a t o m s of t h i o s u l f a t e (S-SO32-) to sulfate,

t h e m e c h a n i s m of o x i d a t i o n of t h e o u t e r sulfur a t o m (S-) has n e v e r b e e n f u l l y ex- plained1, 2

O u r s t u d y on t h e 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 3 led to t h e i s o l a t i o n o f a n e n z y m e which o x i d i z e d e l e m e n t a l sulfur to

t h i o s u l f a t e w i t h G S H as c o f a c t o r :

Biochim Biophys Acta, i22 (1966) 22-33

SULFUR-OXIDIZING ENZYME OF THIOBACILLI 23

The enzyme was tentatively identified as an oxygenase from the results of 180z- experiments 4

In this report we present evidence that Eqn 3 is actually a sum of Eqns I and 2 and sulfite rather than thiosulfate is the initial oxidation product of sulfur as was suggested previously 3 Since the reaction of Eqn 2 was very rapid under ex- perimental 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 formaldehyde- bisulfite complex and determined after hydrolysis of the complex with alkali Cell-free extracts of Thiobacillus thioparus grown on thiosulfate 3 also catalyzed the reaction of Eqn 3 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 thio- sulfate

MATERIALS AND METHODS

Organism

T thiooxidans (ATCC 8o85) was grown as described previously 3 T thioparus

(ATCC 8158 ) was grown in 15-I carboys with aeration Each carboy contained IO 1

of the Starkey's medium No 2 (ref 5): io g Na~S20 a.5H20, 4 ° g K H z P Q , 4 ° g K~HPO4, 0-7 g CaC12, 3 g (NH4)~SO4, 0.2 g FeCI~.6H20, 0.2 g MnSO4.2H~O, 3 ml phenol red (2%) and distilled water to make up a total volume of io 1 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 p H 7 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 3 T thioparus cells were suspended in 0.2 M Tris-HC1 buffer of p H 7.4 (I5%, w/v) and sonicated in a

Io kcyles Raytheon oscillator for 20 min under N 2 atmosphere The cell-flee extract was obtained after removal of cell debris by centrifugation at 23 500 × g for 20 min

Purification of the enzyme

The sulfur-oxidizing enzyme of T thiooxidans was purified as described pre- viously ~ 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 concentra- tions of 15% and 30% after overnight incubation at IO ° The enzyme of T thio- parus was purified b y the same procedures

Further purification was achieved b y 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 p H 7.5 (6 mg enzyme protein/ml) was stirred for 30 min at 4 ° 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

w a s h e d twice w i t h t h e s a m e v o l u m e of buffer at o.o5 M a n d o I M, r e s p e c t i v e l y The e n z y m e was finally e l u t e d f r o m the cellulose w i t h 0.25 M Tris-HC1 (pH 7.5)

P r o t e i n was d e t e r m i n e d b y t h e 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 t h e e n z y m e was followed m a n o m e t r i c a l l y a t 3 °° in

a W a r b u r g a p p a r a t u s The r e a c t i o n m i x t u r e c o n t a i n e d , unless otherwise i n d i c a t e d ,

500 # m o l e s of Tris-HC1 (pH 7.8), 48 m g of sulfur, 5 / , m o l e s of G S H , 250 # g of c a t a l a s e (EC I I I I 6 ) , 0.2 ~ m o l e of 2 , 2 ' - d i p y r i d y l , e n z y m e (0.6 m g of t h e 3 o % e t h a n o l pre-

c i p i t a t e ) a n d w a t e r to m a k e a t o t a l v o l u m e of 2.0 ml The r e a c t i o n was s t a r t e d b y the a d d i t i o n of G S H A f t e r 21o rain t h i o s u l f a t e was d e t e r m i n e d b y t h e m e t h o d of SORBO 7 as d e s c r i b e d a

Determination of thiosulfate and sulfite in the initial product experiments

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 two i n d e p e n d e n t procedures The m e t h o d A was a m o d i f i c a t i o n of t h a t of GOLDMAN AND YAGODA 8 w h e r e

t h i o s n l f a t e was t i t r a t e d w i t h iodine in t h e presence of f o r m a l d e h y d e , a n d sulfite was

t h e n t i t r a t e d w i t h iodine a f t e r t h e d i s s o c i a t i o n of t h e f o r m a l d e h y d e bisulfite c o m p l e x

w i t h s o d i u m c a r b o n a t e , i ml of t h e r e a c t i o n m i x t u r e was t r e a t e d w i t h I ml of

u r a n y l a c e t a t e (0.8%) to r e m o v e p r o t e i n s a n d GSSG To t h e s u p e r n a t a n t a f t e r centri-

f u g a t i o n o.I ml of 4 0 % f o r m a l d e h y d e was a d d e d a n d a f t e r 5 rain t h e m i x t u r e was

t i t r a t e d w i t h O.Ol N iodine to a b l u e end p o i n t w i t h a s t a r c h i n d i c a t o r 5 ml of

s o d i u m c a r b o n a t e buffer (80 g s o d i u m c a r b o n a t e d i s s o l v e d in 500 ml w a t e r , a f t e r

a d d i t i o n of 20 ml of glacial acetic a c i d d i l u t e d to I 1) were t h e n a d d e d a n d t h e solu-

t i o n was t i t r a t e d a g a i n t o t h e end p o i n t w i t h iodine The first t i t r a t i o n v a l u e g a v e

t h e a m o u n t of t h i o s u l f a t e a n d t h e s e c o n d t h e a m o u n t of sulfite

I n t h e m e t h o d B t h i o s u l f a t e was d e t e r m i n e d a c c o r d i n g t o SORBO 7 as d e s c r i b e d

p r e v i o u s l y 3 a n d sulfite b v a m o d i f i c a t i o n of t h e m e t h o d u s e d b y TRfflPER AND SCHLEGEL 9 m e a s u r i n g t h e 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

o r d e r to a v o i d interference b y f o r m a l d e h y d e p r e s e n t in r e a c t i o n m i x t u r e s , s a m p l e s for a n a l y s i s were frozen at 2o ° for s e v e r a l hours before t h e d e t e r m i n a t i o n s in t h e

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 pre-

v e n t i n g its i n t e r f e r i n g w i t h 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

s a m p l e c o n t a i n i n g o.I to 0.3 # m o l e o f sulfite was t r e a t e d w i t h I ml of I °/o zinc a c e t a t e

a n d was centrifuged To t h e s u p e r n a t a n t o.I m l of I M N a O H was a d d e d a n d t h e

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 t h e n m a d e u p t o 4 ml w i t h w a t e r a n d 0.5 ml of a fuchsin solution (4 ° m g fuehsin in IOO ml of 12.5% H2SO4) was a d d e d , followed, a f t e r IO rain,

b y 0.05 ml of 4 0 % 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 m i n t h e 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 S P 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 w i t h

k n o w n a m o u n t s of sulfite were a l w a y s c a r r i e d o u t s i m u l t a n e o u s l y

Determination of iron, copper and labile sulfide

N o n - h e i n e iron in t h e e n z y m e 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 t h e ferrous 2 , 2 ' - d i p y r i d y l c o m p l e x b y a m o d i f i c a t i o n 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 s a m p l e was t r e a t e d

w i t h 0.05 ml of 5 0 % t r i e h l o r o a e e t i c a c i d to release t h e iron from t h e e n z y m e To t h e

Biochim Biophys Acta, 122 (1966) 22-33

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 b y the enzyme preparation was measured at 325 m# and compared to standards of i, 3, 6 and 8 parts per million copper

Labile sulfide was determined b y a modification of the method of FOGO AND POPOWSKY 12 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 0 5 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,

2 times crystallized), GSH, F A D and FMN, Sigma Chemical Co ; sodium diethyl- dithiocarbamate, 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 sonica- tion for 0 5 h of a sulfur suspension in water containing 0.05% Tween-8o After sonication the sulfur suspension was extensively dialysed to remove any contami- nating metal ions

Metals used for the inhibition studies were: Fe(NH4)2(SO4) 2, FeClu, CuSQ, CoC12, ZnSO 4, MgSO 4 and MnSO 4

T A B L E I

PURIFICATION OF THE SULFUR-OXIDIZING ENZYME FROM T thioparvts

T h e e n z y m e a c t i v i t y w a s 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 T h e a m o u n t of p r o t e i n u s e d

w a s as f o l l o w s : 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 p r e c i p i -

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 T r i s - H C 1 ( p H 7.5), 0.4 m g

protein activity ~ (rag)

Cell-free e x t r a c t 2650 0.43

p H 5.0 s u p e r n a t a n t 188 16.6

3O~o e t h a n o l p r e c i p i t a t e 15o 34.2

D E A E - c e l l u l o s e f r a c t i o n i o o 53-5

* Specfic a c t i v i t y w a s 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 p r o t e i n

RESULTS

Enzyme purification and stability

Results of purification of the sulfur-oxidizing enzyme from T thioparus are given in Table I

Biochim Biophys Acta,

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 purifi- fication was consistent

All fractions could be stored at 20 ° at pH 7.5-8.0 for at least 2 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 7 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 4 ° overnight

Half the protein could be removed by heating the 15 30% ethanol precipitate fraction at 5 °0 for 5 min 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

5 min 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 con- siderable 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 pro- tecting GSH from destruction during incubations Since the enzyme preparation (30 % ethanol precipitate) contained iron as observed by the development of a red iron- dipyridyl 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'-dipyri- dyl 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 b y EDTA was

Biochim Biophys Acta, 122 (1966) 22-33

35

3 0

25

.J

~ zo

5

0

60

S z 0~- ,~ MOLES ~ , ~ 3 4 8

3 2 4

214

120 180 2 4 0 3 0 0

~IME OF tNCUSATION (MINUTES)

F i g I E f f e c t o f 2 , 2 ' - d i p y r i d y l 2 , 2 ' - 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 i n d i c a t e d A t 21o m i n G S H ( 5 / ~ 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

p r o b a b l y due t o t h e s t i m u l a t o r y a c t i o n on t h e o x i d a t i o n of sulfidel4, z5 a n d was

p o s s i b l y c a u s e d b y t h e r e m o v a l of G S H f r o m t h e s y s t e m a t t h e level of g l u t a t h i o n e

p o l y s u l f i d e 3 E D T A d i d n o t cause t h e i n a c t i v a t i o n of e n z y m e , 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 t h e i n i t i a l r a p i d r a t e of o x i d a t i o n T h e 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 T h e i n h i b i t i o n was n o t r e v e r s e d b y t h e a d d i t i o n

of Cu 2+ ions

Zn 2+, Co 2+ a n d Cu 2+ ions a t 10 -4 M were f o u n d i n h i b i t o r y e i t h e r in t h e pres- ence or a b s e n c e of 10 -4 M 2 , 2 ' - d i p y r i d y l T h e degree of i n h i b i t i o n v a r i e d a m o n g

3C

2~

!,o

o

5

0

S 2 0 ~ -

MOLES

O- Pt4I~IANI~ROUtE 2:3.7 :2¢- O~PYRIOYL " 2 3 7

NO CHELATING AGENT 20.0

SODIUM OtETHYL - 12.8

ITHIO CARBAMATE

6 0 IZ'O 180 2110

TIME OF INCUBATION (MINUTES)

F i g 2 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 A l l 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

3C

3

2"

5

240 300

TiME O F I N C U B A T f O N (MIN)

Fig 3 R e v e r s a l of H202 i n h i b i t i o n b y 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 u n l e s s 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 / 2 m o l e s of H202,

25 ° / ~ g of c a t a l a s e or 5 / * m o l e s of G S H were a d d e d 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 m i n

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 2 0 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 G S H a d d e d a t 9o a n d 12o m i n , 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 w e r e

c o r r e c t e d for t h e a m o u n t of o x y g e n e v o l v e d f r o m H~O 2

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

m a y compete with iron on the enzyme and thus cause inhibition Such a competition was reported on the Zn e~ inhibition o f p - h y d r o x y p y r u v a t e 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 3 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 3 the inhibition by H202 was reversed after subsequent addition of catalase and GSH From these results it is clear that

H 2 0 2 destroyed GSH, but not the enzyme Catalase, therefore, probably protects GSH from conversion to GSSG by H202 produced non-enzymically during the oxi- dation 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,

SULFUR-OXIDIZING ENZYME OF THIOBACILLI 29

S 0 2-

,4 / / " _.~.~ ~ J ' J I

5f / j ~ / / - ; ~ os - ~ s,85 |

! ''°/

o 6o ,20 ,~o ~io

TIME OF INCUBATION (MINUTES)

Fig 4 E f f e c t o f G S H c o n c e n t r a t i o n

proceeded linearly for a considerable length of time with a clear dependency on GSH concentrations (Fig 4) In previous experiments 3 the non-enzymatic oxidation

of GSH complicated the results The effect of GSH concentration on the sulfur oxida- tion gave a simple, linear LINEWEAVER-BURK plot 18 (Fig 5) and the Km for GSH was calculated as 6.3 mM

02

3 0 2

id

I/EGSH'] {rnM) - I

Fig 5 LINIWEAVER BuRI~ p l o t o f t h e e f f e c t 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

The G S H r e q u i r e m e n t was specific as was the case w i t h the T thiooxidans

e n z y m e 3 Cysteine, 2 - m e r c a p t o e t h a n o l , 2 , 3 - 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 a c i d d i d n o t r e p l a c e G S H in t h e reaction

Identification of iron as co factor

A n 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 p a s s i n g a m i x t u r e of

e n z y m e a n d 2 , 2 ' - d i p y r i d y l (0.I M) t h r o u g h a s m a l l c o l u m n of S e p h a d e x G-25 a l w a y s

r e s u l t e d in a p r e p a r a t i o n which s h o w e d v e r y l i t t l e a c t i v i t y a n d was o n l y s l i g h t l y

s t i m u l a t e d w i t h 10 4 or I0 -~ M F e 2+ ions R e i s o l a t i o n of t h e e n z y m e f r o m t h e m i x -

t u r e w i t h a b a t c h w i s e D E A E - c e l l u l o s e t r e a t m e n t as d e s c r i b e d 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 5 0 % of t h e original a c t i v i t y a n d was s t i m u l a t e d s l i g h t l y b y F e 2+ ions a t IO 5 M as shown in T a b l e I I F e 2+ ions a t

lO -4 M were i n h i b i t o r y A l t h o u g h t h e s t i m u l a t i o n was n o t 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 o r d e r to e l i m i n a t e t h e p o s s i b i l i t y t h a t c o p p e r was a cofactor, a s i m i l a r

t r e a t m e n t of t h e e n z y m e 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 w i t h D E A E - cellulose was c a r r i e d out The a c t i v i t y of t h e e n z y m e t h u s t r e a t e d was n o t s t i m u l a t e d

b y Cu e+ ions a t lO -5 or IO 4 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 a t IO -~ or

I O - 5 M

F i n a l l y , t h e c o n t e n t 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 frac- tion) 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 t h e r e d u c e d ferrous s t a t e The e n z y m e also c o n t a i n e d

o o i 8 / , m o l e of labile sulfide p e r m g p r o t e i n The 3 0 % 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

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 p e r m g protein The l a b i l e sulfide c o n t e n t was o.o16 # m o l e p e r mg The c o p p e r c o n t e n t of t h e e n z y m e was d e t e r -

m i n e d to be less t h a n 0.002/~mole p e r rag The s p e c t r u m of t h e D E A E f r a c t i o n

s h o w e d one a b s o r p t i o n p e a k o n l y a t 275 m/~ in t h e u l t r a v i o l e t r a n g e a n d no a b s o r p - tion in t h e visible range F M N a n d F A D were i n h i b i t o r y to t h e o x i d a t i o n of sulfur

in a g r e e m e n t w i t h t h e p r o p e r t y of the T thiooxidans e n z y m e 3 A t e b r i n h a d v e r y

l i t t l e 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 i t is u n l i k e l y t h a t t h e e n z y m e con-

t a i n s a n y flavin n u c l e o t i d e s or heine c o m p o u n d s as cofaetor

T A B L E I I

E F F E C T O F I R O N O N T H E P A R T I A L L Y I R O N - F R E E E N Z Y M E

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"-

d i p y r i d y l w a s o m i t t e d a n d f e r r o u s a m m o n i u m s u l f a t e w a s a d d e d t o t h e c o n c e n t r a t i o n i n d i c a t e d

E a c h f l a s k c o n t a i n e d o 4 m g o f t h e e n z y m e t r e a t e d a s f o l l o w s : t h e 3 ° ~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 m l ) w a s t r e a t e d w i t h 2 , 2 " - d i p y r i d y l a t a f i n a l 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 4 °, 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 o 5 M a n d o i M T r i s - H C 1 ( 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 02 uptake S20a 2-

of Fe 2+ (l~moles) formation

(#moles)

Biochim Biophys 4cta, 122 (1966) 2 2 - 3 3

SULFUR-OXIDIZING ENZYME OF T H I O B A C I L L I 31

T A B L E I I I

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 2 m l : 5 0 0 / * m o l e s T r i s - H C 1 ( p H 7.8), 25 ° # g

c a t a l a s e , 0 2 / z m o l e s 2 , 2 " - d i p y r i d y l ; 3.0 m g of T thioparus e n z y m e or 2 5 m g of T thiooxidans

e n z y m e ( 3 0 % e t h a n o l p r e c i p i t a t e ) , 48 m g s u l f u r , 5 # m o l e s G S H , 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

T h e i n c u b a t i o n w a s c a r r i e d o u t a t 3 °0 for 21o m i n in air A, B: T h i o s u l f a t e a n d s u l f i t e w e r e

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

T thioparus enzyme

S + G S H + H C H O (IO # m o l e s ) 26.5 2o.o 21.8 5-5 5 8

S + G S H + H C H O (5 ° # m o l e s ) 25.6 13.3 15.9 lO.5 12-3

S + G S H + H C H O ( i o o # m o l e s ) 22.4 8.0 8 7 14 7 16 4

S + G S H + H C H O (2o0 # m o l e s ) 13 7 1.6 1 4 13.5 13.6

Na~S203 (20 # m o l e s ) + G S H 2.5 19.4 19.2 0.5 0.5 NaS208 (20 # m o l e s ) + G S H + H C H O

T thiooxidans enzyme

S + G S H + H C H O (5o # m o l e s ) 11.4 8.8 7.0 2.6 2.2

S + G S H + H C H O ( i o o # m o l e s ) lO 7 6.6 5.3 5.2 4.6

Initial product of sulfur oxidation

As shown in Table I I I formaldehyde trapped sulfite during the oxidation of

came more efficient with higher concentrations of formaldehyde The enzyme activity was not inhibited appreciably b y formaldehyde below 0.05 M as manifested b y 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 3

T A B L E I V

NON-ENZYMIC REACTIONS OF SULFUR COMPOUNDS

T h e c o n d i t i o n s w e r e 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

S + G S H + H C H O (5o # m o l e s ) 0 4 0.8 0.8 i o i i Na2SaO 3 (20 # m o l e s ) + G S H 1 5 2o.0 19.4 0.5 0.5 Na~S20 s (20 # m o l e s ) + G S H + H C H O

S + N a , S O 8 ( i o # m o l e s ) + G S H lO.4 9.6 o.o o.o

Biochim Biophys Acta,