Iron-containing reductases: Investigation of sirohaem-dependent reactions involving sulphur oxyanions in desulphoviridin from Desulphovibrio gigas—I Characterisation of physical and enzymic properties of desulphoviridin

J. inorg, nucL Chem, Vol. 43. pp. 815-823, 1981 Printed in Great Britain

0022-19021811040815419502.0010 Pergamon Press Ltd.

BIO-INORGANIC SECTION IRON-CONTAINING REDUCTASES: INVESTIGATION OF SIROHAEM-DEPENDENT REACTIONS INVOLVING SULPHUR OXYANIONS IN DESULPHOVIRIDIN FROM DESULPHO VIBRIO GIGASmI C H A R A C T E R I S A T I O N OF P H Y S I C A L AND ENZYMIC PROPERTIES OF DESULPHOVIRIDIN. M. HELEN HALL and REG H. PRINCE University Chemical Laboratory,Lensfield Road, Cambridge, England

(Received 1 March 1980;receivedfor publication I July 1980) Abstract--The physical and enzymic properties of desulphoviridin from Desulphovibriogigas have been characterised for reactivity studies. The prosthetic groups were identified as 2 sirohaems and groups derived from 8 Fe/S per molecule of a.,/~2 type. On polyacrylamidegel electrophoresis, native desulphoviridin exhibited two bands which are thought to be isozymeswith the same subunit structure but possibly carryinga different charge. INTRODUCTION

Sulphite reductase enzymes catalyse the six-electron reduction of sulphite to sulphide for the purpose of either sulphur assimilation, or as part of the reduction of sulphate performed for the release of energy by the bacteria Desulphovibrio and Desulphotomaculum [1]. The assimilatory sulphite reductases (E.C. 1.8.1.2.) of E. coil and S. typhimurium have been well characterised (mainly through the work of Siegel et al. ([2] and refs. therein]) with respect to the number and type of prosthetic groups and the electron transfer sequence through them[31. Such detail is not known for the dissimilatory sulphite reductases. It has been shown that the same prosthetic group, sirohaem, is shared by both the assimilatory and dissimilatory sulphite reductases[4]. Sirohaem is an iron porphyrin of an unusual type, postulated by Murphy et a/.[5] to be an iron-containing methylated tetrahydroporphyrin of the isobacteriochlorin type, although the release and identification of the prosthetic group from D. gigas desulphoviridin by Battersby et al.[6] has shown it to be mostly in the form of the mono-lactone. As the prosthetic group of D. gigas desulphoviridin has been released only as the metal-free porphyrin, sirohydrochlorin[6--8] it has been suggested that the FeN bond length is unusually long in this instance. Sirohydrochlorin alone will catalyse the reduction of dithionite to sulphide [9, 10]. Dissimilatory sulphite reductases (E.C. 1.8.7.1.) can be divided into three groups of which the best-studied is desulphoviridin, the green enzyme found in all species of Desulphovibrio except one. Nevertheless, little is known *Abbreviations used in the experimental section are as follows. tris = tris(hydroxymethyl)aminomethane; EDTA= 1,2-diaminoethane-N,N,N',N'-tetraacetic acid or anion; MVH reduced methylviologen; SDS = sodium dodec-¢l sulphate; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotide; NAD(P)= nicotinamide adenine dinucleotide(phosphate); YAD = yeast alcohol dehydrogenase; HCAB=human carbonic anhydrase, isozyme B; DSV = desulphoviridin: Temed= N,N,N',N'-tetramethyl-1,2-diaminoethane. JINC Vol. 43, No. 4.--L

concerning their mechanism of action, although D. vulgaris desulphoviridin has been partially characterised[7, 11, 12]. A study was therefore undertaken of desulphoviridin purified from D. gigas[12c]. This paper describes the physical characterisation of the enzyme in order to ascertain the number and nature of the prosthetic groups. The enzymic properties are also investigated, in particular the function of the two bands exhibited by native desulphoviridin on polyacrylamide gel electrophoresis (PAGE). EXPERIMENTAL*

All manipulationsof enzymesolutions were carried out at 4°C under oxygen-freenitrogen or argon. De-ionizedwater was used throughout. PURIFICATION OF DESULPHOVIRIDIN

(i) Growth o[ Desulphovibrio gigas Desulphovibrio gigas bacteria were obtained as a lyophilized sample from the National Collection of Industrial Bacteria, Tory Research Station, Aberdeen; strain No. 9332. Revival of the culture was carried out using the procedure recommended by the NCIB. The following growth media were used [12a, b]: (a) Basal medium: K2HPO4 (0.5g), NH4CI (1.0g), Na2SO4 (1.0 g), CaCI2.2H20 (0.1 g), MgSO4'7H20 (2.0 g), sodium lactate (5.0 cm3, 70% w/w solution), yeast extract (l.0g) and deionised water (I 13. This medium was adjusted to pH 7.4 with aqueous 2 M KOH and distributed in 200cm 3 amounts in screw-capped bottles and autoclaved at 125°C for 30 rain. (b) Complete medium: To each 200 cm3 bottle of basal medium were then added thioglycollic acid (0.015 cm3), ascorbic acid (20 mg) and FeSO4.7H20 (100 rag). The pH was then adjusted to 8.1 with 2 M KOH. Once the organisms were established in the log phase, they were transferred to the basal medium. Immediately before inoculation 2-mercaptoethanol (0.01%v/v) was added to the medium. Anaerobicity was maintained as before, and the bacteria were transferred to fresh growth medium every 3 days using a 10% iv/v) inoculum. The 815

M. H. HALL and R. H. PRINCE

816

incubation temperature was 32°C. The advantage of using the basal medium is that large quantities of Fe(II) sulphide are not produced during growth, which simplifies the isolation of the cells. For each preparation of desulphoviridin, D. gigas cells were grown in 25-1. carboys, completely filled and sealed. Each carboy was inoculated with ca. 3 I. of cell culture. (ii) Harvesting o[ cells D. gigas ceils were collected by centrifugation in a Sharpies centrifuge at a rate of 100cm3/min. For each preparation of desulphoviridin, 25 I. of growth medium yielded approximately 30g wet weight of cells. If not used immediately, the cells were stored at -20°C for no longer than six months. (iii) Preparation o[ cell extract The bacterial cells were ruptured by sonication. The subsequent purification is based on the method of Jones and Skyring[13]. The pellet of harvested cells was suspended in 30 cm3 of 1 mM EDTA-0.1 M tris/HCI-0.1 M KCI buffer, pH7.8 and sonicated at the maximum frequency in a Dawe soniprobe (type 7530 A) for a total of 5 rain. The solution was then centrifuged at 17,000rpm for l hr. The supernatant liquid was retained at 4°C and the pellet resuspended in 20 cm3 of the same buffer and sonicated as before for a further 5 min. Separation of the supernatant from the cell membranes was again by centrifugation at 17,000rpm for I hr. The total volume of the supernatant was approx. 60 cm3. The absorption spectrum of this fraction is dominated by large absorption maxima at 280 and 260 nm, but peaks at 410 and 627 nm can be seen. The sulphite reductase activity at this stage could not be measured accurately because of interference by other proteins. (iv) DEAE-52 chromatography The crude sonicate was loaded onto a column (45 × 2.5 cm) of DEAE-52 resin (Whatman) previously equilibrated with l mM E D T A ~ . i M tris/HCI--0.1 M KCI buffer, pH7.8, at a flow rate of 60cm3/hr. Washing continued with this buffer and at this flow rate until no more protein was eluted. All protein estimations during the purification were by the method of Christian and Warburg[14]. More protein was then eluted from this column with the second wash of 1 mM EDTA---0.1 M tris/HCI--0.2 M KCI buffer, pH 7.8, and again this wash was continued until no more protein was eluted. Desulphoviridin was eluted with the third wash of 1 mM EDTA--0.1 M tris/HCl, 0.3 M KCI buffer, pH 7.8 and could be seen moving down the column as a green band. Fractions of 5cm 3 were collected and certain fractions pooled after assay of both the protein content and the desulphoviridin content (by estimation of the absorption maximum at 627 nm). The volume of the pooled fractions was reduced by

A

tris buffer tris 0.1M HCI Temed H20

36.3 g 48.0cm 3 0.46 cm~ to 100 cm3

dialysis against solid sucrose to approx. 9 cm3. Sucrose removal was by dialysis against 0.1 M tris/HCI buffer, pH 7.0. The absorption profile of this eluate exhibited maxima at 381, 390, 410, 581 and 630nm. The activity (spectrophotometric assay) was ca. 0.15 ~mole reduced MVH oxidized/rain/0.15 mg. The total desulphoviridin content was 30-40 mg. (v) Sephacryl S-200 chromatography The enzyme purified by chromatography on a DEAE52 column was loaded onto a column (60 x 2.5 cm) of Sephacryl S-200 previously equilibrated with 0.1M tris/HCI buffer, pH 7.0, at a flow rate of 50--60cm~/hr. Sephacryl S-200 gel (Pharmacia Fine Chemicals) was sterilized by autoclaving before use. The desulphoviridin could again be seen moving down the column in a tight green band. Fractions (5cm 3) were collected and the desulphoviridin fractions combined after assaying each fraction for protein and desulphoviridin content; the combined fractions were then reduced in volume by dialysis against solid sucrose, and finally extensively dialysed against 0.1 M tris/HCI buffer, pH7.0. The absorption spectrum of this purified eluate exhibited peaks at 381, 390, 410, 580 and 627 nm, as shown in Fig. 1. The activity (spectrophotometric assay) was ca. 0.1 ttmol MVH oxidised/min/0.15 rag. Typically, the yield of desulphoviridin was approx. 30rag in 10cm 3. An absorbance of 0.16 units at 627nm was found to represent 1 mg desulphoviridin/cm ~ in three separate preparations of the enzyme (path length 10 nm). 2

.g o

0

4C)0

450

5C)0 5~0 )~(nrn)

6(bO

650

Fig. I. Visible absorption spectrum of native desulphoviridin. Path length 10rnm, protein concentration of DSV preparation, 3.2 mgcm-3. POLYACRYLAMIDE GEL ELECTROPHORESIS

The desulphoviridin was shown to be homogeneous by PAGE. A Shandon Southern Analytical PAGE outfit was used, incorporating 12 gel columns of length 8 cm. Gels containing 5% acrylamide were used, prepared from the following solutions;

13

C

Acrylamide Solution

Initiator

Acrylamide Bis K3Fe(CN)6 H20

Ammonium persulphate 0.14 g H20 to I00 cm3

10 g 0.27 g 0.005 g to 100 cm~

Iron-containing reductases BDH chemicals were used and were of Analar grade. The solutions were mixed in the ratio IA: 2B : 4C: 2H20. Polymerisation took approx. 15 min. The composition of the reservoir buffer was glycine, 28.8 g; tris, 6.0 g in water to I I. Desulphoviridin (0.05-0.075 mg) was mixed with concentrated sucrose solution and tracker dye, and layered on top of the gels. The current used was 3 mA/tube. Protein migrated down the gels to the anode and electrophoresis was stopped when the tracker dye had almost reached the bottom of the gel, after approx. 20 rain. Gels were removed from the tubes by the irrigation needle method and were stained for protein with Amido Black (1% w/v in 7% acetic acid); they were destained by electrophoresis. Desulphoviridin from D. gigas subjected to electrophoresis in this way gave two green bands, closely separated. The bands only were stained with the proteinsensitive dye. The faster-moving of the two bands was termed the major band and contained the most protein, and the slower of the two bands was termed the minor band. The relative mobility of each band was calculated from the expression: Relative mobility, Rf

distance of protein migration distance of TD* migration ×

length before staining length after staining

(* Tracker dye) Typically, Rf Imajor band) = 0.14 Rf (minor band)= 0.16 To ensure that both these bands were desulphoviridin the gels were assayed for sulphite reductase activity by a modified method of Skyring and Trudinger[15]. Screwcapped test-tubes of volume 30cm 3 were completely filled with a solution of 0.1 M tris/HCI buffer, pH 8.0 or 7.0, containing 12% MVH and 2 mM FeSO4"7H20, previously deoxygenated under vacuum. The MV was heated with zinc to give the stable violet-blue reduced form. After electrophoresis the gels were transferred to these tubes, 50rag of anhydrous Na2SO3 added, the tubes again closed and shaken to dissolve the added

sulphite and remove any traces of 02 taken in with the gel. The gels were then incubated at 30°C for 2 hr, after which they appeared to be uniformly stained dark-blue by the MVH; excess MVH was removed by standing the gels in distilled water for 3 hr. Both the major and minor bands could then be identified as black bands due to the deposition of FeS resulting from sulphite reductase activity. Both bands could also be recognized as desulphoviridin in the original gels by their UV fluorescence in 2 M NaOH, THE MAJOR AND MINOR BANDS OF DESULPHOVIRIDIN

After removing twelve gels subjected to electrophoresis from their tubes, the major and minor bands from each were sliced from the gels. All the major bands were then suspended in 2 cm 3 of 0.1 M tris/HCl buffer, pH 7.0, the minor bands similarly. The gel sections were comminuted for maximum extraction, and stirred in the buffer solution overnight. Protein was recovered by removing the buffer solution, and reducing the volume by sucrose dialysis to 1 cm 3. The absorption spectra of the major and minor band preparations extracted in this way are shown in Fig. 2: they are identical to the spectrum of S-200-purified desulphoviridin with absorption maxima at 381,390, 410, 580 and 627 rim. Each of these preparations (approx. 0.05 rag) was resubmitted to electrophoresis as before on 5% acrylamide gels, and both showed a single band when stained with Amido Black. For the major band preparation this band corresponded to the original major band with the same Rf value, and the minor band, re-submitted to electrophoresis gave a band which also had the same relative mobility as the original minor band. Also, for each preparation, a very faintly staining band corresponding to the other band could be detected. This second faint band was attributed to contamination of, say, the major band preparation by minor band protein (and vice versa) due to the difficulties of mechanically separating accurately the two green bands in the original gel. Both bands of the major and minor preparations resubmitted to electrophoresis, showed sulphite reductase activity, both in the gels by the deposition of FeS according to the assay described earlier and by the spectrophotometric assay. The activity of the major band preparation was 50l~mol/MVH/min/O.15mg, that of

8

8

4OO

817

450

X(nm) Fig. 2. Visible absorption spectrum of the major and minor band proteins. Both bands have identical spectra. Path length 10 mm, protein concentration, 2.6 mg cm-3.

818

M.H. HALL and R. H. PRINCE

the minor band preparation was, 30#, mol/MVH/min/ 0.15rag. The major and minor bands are not due to different oxidation states of the enzyme. This was shown by incubating equal volumes of desulphoviridin anaerobically with 2 mM dithionite and 2 mM ferricyanide for 5, 10 and 20 min at room temperature, to reduce and oxidise it respectively. The enzyme was then subjected to electrophoresis as usual. Both the reduced and oxidised desulphoviridin showed major and minor bands with the same relative mobilities as the desulphoviridin standard. Nor are the major and minor bands the product of different conformations of the polypeptide chains, at least if the protein conformation depends on disulphide bridges. Incubation of desulphoviridin with Cleland's reagent (Sigma) for 5 or 15 min at room temperature did not alter the electrophoretic behaviour of the protein. POLYACRYLAMIDE GEL ELECTROPHORESIS PRESENCE OF UREA

IN

THE

Dissociation of native desulphoviridin, the major band preparation and the minor band preparation into their component subunits was achieved by incubation with urea. The three protein fractions were each incubated with 0.1 M tris/HCI buffer, pH 7.5 containing !-8 M urea, for 5-7 hr at room temperature, and then subjected to electrophoresis at 3 mA per gel in the 5% acrylamide gel system described previously. The reservoir buffer also contained the appropriate concentration of urea. After electrophoresis, the gels were stained with Amido Black and destained electrophoretically as before. Desulphoviridin, and both the major and minor band preparations showed two bands only under all conditions. The Rf values of these bands decreased as the urea concentration increased. The results are tabulated in Table I. The urea could be removed by extensive dialysis against the original buffer of the enzyme, to restore the original electrophoretic behaviour. This does, however, cause (on average) a 50% loss of activity.

SODIUM DODECYL SULPHATE GEL ELECTROPHORESIS

Further characterisation of desulphoviridin from D.

gigas was by polyacrylamide gel electrophoresis in the presence of SDS by the method of Weber and Osborne[16]. Desulphoviridin showed two bands on SDS gel electrophoresis with relative mobilities of 0.49 and 0.29. Identical bands were produced on SDS gel electrophoresis of the major and minor band preparations. This indicates that native desulphoviridin, the major band preparation and the minor band preparation contained two different polypeptide chains, the same two in each case. It is possible that a second band appeared on the SDS gels because of protease activity, although this is not likely as each protein had been shown to be electrophoretically pure. However, the possibility had to be eliminated. Native desulphoviridin was prepared as usual for electrophoresis and then incubated in a boiling water bath for 2 rain to destroy any protease activity before electrophoresis. Staining showed the original two bands with the same mobilities as the desulphoviridin standard. The RMM's of the two subunits were estimated by SDS gel electrophoresis with the proteins shown in Table 2 as standards: Table 2. The relative molecular masses of proteins used as SDS gel electrophoresis standards RM~*

cyctochrome

c

11,700

RCAB

29,000

YAD

37,000

catalase

60,000

bovine

serum albumin

68,000

B-galaetosidase (*

All

proteins

130,000 supplied

by Sigma)

Table 1. Effect of urea concentration on polyacrylamide gel electrophoresis of desulphoviridin and its constituents

Dsv

Maj

Min

*

lm*

2m

3m

4m

5m

6m

7m

8m

0.38

0.35

0.31

0.28

0.26

0.24

0.23

0.21

0.23

0.28

0.21

0.20

0.26

0.16

0.14

0.14

0.38

0.34

0.31

0.29

0.26

0.24

0.23

0.22

0.23

0.28

0.22

0.20

0.17

0.17

0.15

0.14

0.38

0.35

0.31

0.28

0.26

0.24

0.23

0.21

0.22

0.28

0.22

0.21

0.17

0.17

0.14

0.13

concn,

urea

Maj =major b a n d p r e p n . Min = m i n o r

The figures

band prepn.

given

are

Rf values,

as defined

in

text.

M. H. HALL and R. H. PRINCE Approximately 5 mg of each protein (including native desulphoviridin, the major band preparation and the minor band preparation) were incubated separately in 1 cm3 of the standard incubation buffer for 2 hr at 37°C before SDS gel electrophoresis on 10% acrylamide gels. Four mixtures were also prepared and subjected to electrophoresis: (a) protein standards only; (b) protein standards and native desulphoviridin: (c) protein standards plus the major band preparation; (d) protein standards plus the minor band preparation. After electrophoresis the gels were stained with Coomassie Blue and de-stained as described previously. Figure 3 shows the graph of Rf against log (RMM). From this the RMM's of the two polypeptide chains are 44,600 and 61,000 respectively.

819

190 170 150

I10

x

90

x~, x

4'.1

i

i

i

4.5

4.9

THE RMM OF NATIVE DISULPHOVIRIDIN

RMM 12,400 150,000 275,000 480,000 520,000

YAD

xanthine oxidase apoferritin /3-galactosidase

i

53 i

'

*RMM values from Andrews[17]. All proteins were supplied by Sigma. Sephadex G-200 was purchased from Pharmacia Fine Chemicals. Figure 4 shows the calibration curve of elution volume against log(RMM). The RMM of native desulphoviridin is 210,000 daltons.

5.7 '

Fig. 4. Elutionvolumesof proteins, including DSV, as a function of relative molecular mass. The proteins, in order of increasing molecular mass, are: cytochrome c: YAD; DSV; xanthine oxidase; apoferritin;/3-galactosidase. AMINO ACID ANALYSIS Purified desulphoviridin was subjected to amino acid analysis. The protein was hydrolysed for 24 hrs in 6 M He1 at 105°C in sealed evacuated tubes. The results are shown in Table 3. Three separate attempts at carboxymethylation of desulphoviridin proved unsuccessful, and therefore the number of cysteine residues is unknown. However, the amino acid analysis described above showed a small peak in the cysteine position, and this normally indicates a high proportion of cysteine. This is expected for desulphoviridin. THE pH-RATE PROFILE OF NATIVE DESULPHOVIRIDIN

The pH rate-profile of native desulphoviridin is shown in Fig. 5. The enzyme was assayed for sulphite reductase Table 3. Aminoacid content of desulphoviridin No.

of

residues

Nearest

interger

Asp

152.398

152

Thr

103.474

103

Ser

110.409

II0

Glu

201.698

202

Pro

101.599

102

Gly

180.141

180

Ala

243.500

244

Val

134.216

134

Met

38.053

38

iLeu

102.536

103

Leu

140.402

140

Tyr

49.675

50

Phe

69.36

69

His

42.739

43

Lys

104.411

104

Arg

100.099

i00

x 5.0

2; v

o0, 4.6

x

4.2

~

I 0

i 0.2

I 0.4

0.6

0.8

\

Iog(R M.M)

The RMM of native D. gigas desulphoviridin was estimated by gel filtration. Sephadex G-200 gel filtration medium was allowed to swell in 0.1 M tris/HCI buffer, pH 7.5, for at least three days until it had reached maximum volume, and then packed at a constant flow-rate of 15cm3/hr into a 50× 2.5 cm column. The flow-rate subsequently remained the same. The standard proteins used were:

cytochrome c

i

1.0

Rf

Fig. 3. Proteins used as RMM standards: relation between mobility and relative molecular mass.

820

M.H. HALL and R. H. PRINCE (a) Iron A standard curve was first constructed using solutions of 1-10 pore Fe diluted from a stock solution of 100 ppm Fe. The iron salt used was AR FeSO4.7H20, dissolved in deionised H20 containing 0.05 M HCI. Two different preparations of desulphoviridin were analysed; the results are tabulated below. There was no effect on these results if the desulphoviridin preparations were first treated with 20% trichloroacetic acid, in case protein was masking Fe.

2.0

> 1.0 :<

~ x ~ X ~ x

// ~.o

'

6'.o

'

,!o

'

~.o

'

pH

Fig. 5. Effect of pH on the activity of native desulphoviridin (activity=# mole MVH oxidised per minute per 0.15rag of reductase). Solutions are in closed 10mm quartz cells with deoxygenated nitrogen circulation). activity spectrophotometrically[13] and the oH of the pigment solution altered by dialysis against 0.1 M tris/HCI buffer adjusted to the required pH. THE TEMPERATURERATE-PROFILEOF NATIVE DESULPHOVIRIDIN The activity of desulphoviridin was measured by spectrophotometric assay at temperatures from 20°C to 50°C, Fig. 6; maximum activity occurs at 37°C. FLAVINANALYSISOF DESULPHOVIRIDIN Desulphoviridin, on the basis of spectrophotometric properties alone, would appear not to contain flavins, but this was investigated more precisely by the riboflavin fluorescence method/18]. Riboflavin, FMN and FAD obtained from BDH were used as standards. Desulphoviridin was found to contain no flavin component. REDUCTIONOF DESULPHOVIRIDINBY NADPHAND FLAVINS Desulphoviridin was unable to use flavins and/or NADPH as a reductant for sulphite reductase activity as shown by substituting these reagents for MVH in both the gel incubation and the spectrophotometric assays. ESTIMATIONOF THE METALCONTENTOF DESULPHOVIRIDINBY ATOMICABSORPTION SPECTROPHOTOMETRY

Atomic absorption (AA) spectrophotometry was used for the estimation of various metals in desulphoviridin with due regard to the removal of any possible masking agents. A Pye Unicam SP 90B AA spectrophotometer was used with an air/acetylene flame except for Mo which required a nitrous oxide/acetylene flame.

:>

Nx

o

/ i

./ i

20

i

/

BO

i

i

40

I

Fig. 6. Effect of temperature on the activity of native desulphoviridin (activity is expressed as in Fig. 5).

dsv (i) dsv (ii)

Protein concn. (mg/cm3)

Fe content (ppm)

Fe content (atoms/tooldsv)

2.5 2.7

7.0 7.05

10 9.7

Desulphoviridin therefore contains 10 Fe/mol. (b) Other metals Solutions ranging from 1 to 10ppm were used to calibrate the spectrophotometer for each of the other metals. BDH Analar salts were used. Comparison with two preparations of desulphoviridin showed that this protein contained no Cu, Mn, Zn, Mo or Co. ESTIMATIONOF HAEMIN DESULPHOVIRIDIN The isolation of the prosthetic group was by methanolH2SO4 extraction/6/. Desulphoviridin (135.16 rag) yielded 1.07 mg of prosthetic group by this purification procedure. There are therefore 1.7mole of prosthetic group/mole of desulphoviridin. ESTIMATIONOF NON-HAEM IRONIN DESULPHOVIRIDIN The non-haem iron content of desulphoviridin was determined using an adaptation of the method of Harvey et a/.[19] based on complex formation with 1,10 phenanthroline. Two separate preparations of native desulphoviridin were examined in this way. Comparison with the standard curve indicated 8.2 and 8.9 Fe/molecule of desulphoviridin. Cytochrome c was also examined by this method to ensure that haem Fe is masked from the reaction, and indeed no iron was detected in this protein by this method. ESTIMATIONOF THE INORGANICSULPHIDEIN DESULPHOVERIDIN The quantity of inorganic sulphide in desulphoviridin was estimated by the method of Gilboa-Garber[20], based on incorporation into methylene blue. Two desulphoviridin preparations were examined and comparison with a standard curve indicated 8.4 and 8.7 S/molecule. Cysteine, glutathione, insulin and bovine serum albumin did not react in this test, confirming that this method is specific for inorganic sulphide. SPECTROPHOTOMETRICPROPERTIESOF DESULPHOVIRIDIN Native desulphoviridin obtained from D. gigas by the purification procedure described earlier exhibited absorption maxima at 381, 390, 410, 580 and 627 nm. All spectra were recorded under strictly anaerobic conditions using a Pye Unicam SP800 spectrophotometer. Typically, 10#1 of the reagent was added to desul-

Iron-containingreductases phoviridin to give a final concentration of 10 mM. The enzyme concentration was 5 raM. Incubation of desulphoviridin with its substrate sulphite, for as long as 2 hr at room temperature, produced no change in its absorption spectrum. There was also no change on addition of NOC, NH2OH, CN-, $2032- and $3062-. No detectable complex is formed with CO. The addition of a small amount of dithionite, <5 raM, produced a decrease in the absorption of the whole spectrum, but no shifts in any peaks. This decrease is most pronounced in the region around 450 nm. There was no further change after incubation at room temperature for 20 rain. The original spectrum is restored by oxidation in air or by the addition of ferricyanide. Desalting the enzyme also restores the original spectrum; desulphoviridin and dithionite passed down a column (10× 1.5 cm) of Sephadex G25 resin (Pharmacia), previously equilibrated with 0.1M tris/HCI buffer, pH7.0, apparently removed all dithionite and ferricyanide from the pigment as the original behaviour was restored. If excess dithionite is added to desuiphoviridin the peak at 580nm disappears and slight peaks at 550 and 500 nm are formed. This can be seen in Fig. 7. Again, there was bleaching below ca. 520 nm, and this is most pronounced between 450-460 nm. The peaks at 410 and 627 nm are decreased slightly in intensity but not shifted. Oxidation by any of the three methods described earlier restores the original spectrum. The difference spectrum of desulphoviridin vs desulphoviridin plus excess dithionite shows only one minimum at 450 nm. In the presence of both excess dithionite and KCN the bleaching around 460 nm is even greater. The decrease in the peak at 410 nm is also greater, and the appearance of the peak at 500 nm, slight with dithionite alone, becomes more evident. The optical spectrum of desulphoviridin in the presence of reduced MV yields no information because of the very large and broad absorption of MVH centered at 600 nm, obscuring the appropriate part of the desulphoviridin spectrum. The difference spectrum of desulphoviridin vs desulphoviridin plus MVH is similar to that for desulphoviridin plus dithionite and shows a minimum at 450nm, but also a shift of the a-peak to higher wavelengths.

821 DESULPHOVIBRIOSPECIES 10455

The desulphoviridin from D. sp 10455 was purified for comparison with the enzyme from D. gigas. (i) Growth of D. sp /0455 D. sp 10455 were obtained as a lyophilised sample from the National Collection of Industrial Bacteria, strain No. 10455. The bacteria were grown following the procedure for 19. gigas, using Starkey's medium (12b) and incubating at 30°C. (ii) Harvesting o[ cells The same procedure was used as for D. gigas: net wet weight of cells obtained ca. 20 g. PREPARATION OF DESULPHOVIRIDIN

The conditions for this preparation were exactly those of the preparation of desulphoviridin from D. gigas: the yield of desulphoviridin from 19. sp 10455 was ca. 20 rag. Spectra of the D. sp 10455 after DEAE-52 and Sephacryl S-200 chromatography were practically the same as those of the D. gigas pigment. ELECTROPHORETICPROPERTIES

Polyacrylamide gel electrophoresis of desulphoviridin from D. sp 10455 on 5% acrylamide gels gave one band. Conditions for electrophoresis were the same as those used for the D. gigas enzyme. The relative mobility of this single band was 0.14, the same as the major band of D. gigas desulphoviridin. The band also exhibited sulphite reductase activity on incubating the gel with sulphite and MVH as for D. gigas. SDS gel electrophoresis showed two components, again as for the D. gigas enzyme, with molecular weights of 44,500 and 60,000. The elution volume of 19. sp 10455 desulphoviridin on a Sephadex G-200 solumn, 50 × 2.5 cm, equilibrated with 0.1M tris/HCI buffer pH7.5, was ll5cm 3. giving an estimated R.M.M. of 210,000 daltons. OPTICAL SPECTRA

Sephacryl S-200--purified desulphoviridin from D. sp 10455 gave a spectrum closely similar to that of the D. gigas pigment with peaks at 632, 583, 411 and 391 nm. This spectrum changed only on the addition of either dithionite or MVH as found for desulphoviridin from D. gigas. DISCUSSION

0

400

4,50

500

550

600

h(nm)

Fig. 7. Effect of dithionite on the absorption spectrum of native desulphoviridin (see text): --, DSV ....... DSV+ excess $2042 .

tThis agrees with the spectra of desulphoviridin previously purified from D. gigas[131and D. vulgaris[I II.

We have purified desulphoviridin from O. gigas to homogeneity, as shown by polyacrylamide gel electrophoresis. The yield of enzyme is typically 20% of sonicated protein. The absorption spectrum of the enzyme exhibits maxima at 381, 390, 410, 580 and 627nm, the peak at 627nm being characteristic of desulphoviridin and having a height of 0.16 absorbance units if the enzyme concentration is 1 mg/cm3 and the path length 10mm.t The activity of the purified desulphoviridin is typically 100 nmoles MVH oxidised/min/0.15 mg, spectrophotometrically assayed. Polyacrylamide gel electrophoresis of native desulphoviridin shows that the enzyme is composed of two components, the major and minor bands, both of which exhibit sulphite reductase activity. The proteins comprising the major and minor bands can be extracted and re-submitted to electrophoresis; they both have ab-

822

M. H. HALL and R. H. PRINCE

sorption spectra identical to native desulphoviridin.:~ In contrast to the results of Jones and Skyring[13], reelectrophoresis of the major and minor band preparations shows that each is a homogeneous protein. The major and minor bands do not represent different oxidation states of the enzymes, nor are they caused by the presence or absence of disulphide bridges. SDS gel electrophoresis reveals that for native desulphoviridin the major and the minor band preparations are each composed of 2 subunits of R.M,M. 44,600 and 61,000 daltons. As the R.M.M. of native desulphoviridin is estimated as 210,000 daltons by Sephadex G-200 gel filtration, these subunits are most likely to be arranged in an a2/~2 structure.§ There are at least two possibilities for the origin of the major and minor bands of desulphoviridin: (a) Each is an !a/~l unit, with a different charge on each, the two together forming the complete a2f12 structure of desulphoviridin. This supposes that the attraction between the two la/~l units is relatively weak, and that the attraction between the a and fl chains within these units is stronger. (b) The major and minor bands could represent different forms of desulphoviridin, both having an az/~z structure but with a different charge. Dissociation of the three forms of the enzyme with various concentrations of urea indicate the second possibility as the most likely. Following urea incubation, each of the three fractions exhibited two bands on 5% acrylamide gels. If desulphoviridin were composed of 21a/31 units which give rise to the major and minor bands on electrophoresis we would expect, at low urea concentrations, to see two bands due to these units, and then at higher concentrations two bands of different mobility due to the a and/~ chains. We would also expect to see one band for each of the major and minor band preparations following incubation at low urea concentrations and two bands at higher urea concentrations indicating the a and /~ subunits. However, all three fractions exhibited two bands at all concentrations of urea, and although the relative mobilities of these bands did alter with varying urea concentrations, they did so gradually. This behaviour was identical for all three fractions and indicates that they all have the same composition, an a2B2 structure. This would not be true if 8 M urea were not capable of dissociating the a and /3 subunits. The major and minor band preparations have identical kinetic properties to those of native desulphoviridin (see following paper in this series). Also, the behaviour of desulphoviridin from D. gigas is the same in many respects (viz: in all properties tested here), to that of desulphoviridin from D. sp 10455, which exhibits only one band on polyacrylamide gel electrophoresis, Therefore the major and minor bands may well represent two forms of native desulphoviridin with slightly different charges. ~The D. gigas enzyme purified by Jones and Skyring[13] exhibited a total of eleven bands on electrophoresis, only six of which were identified as desulphoviridin by their fluorescence in NaOH under UV light. The major and minor bands of native desulphoviridinhave been reported for the enzyme from D. gigas (13%) and D. Vulgaris[21] only. §The molecular weights of the desulphoviddin from D. gigas and D. vulgaris have been reported as 200,000113] and 226,000[7, 11], and those of the D. vulgaris enzyme a and /3 subunits 42.-45,000and 50-55,000 respectively[7].

Kobayashi et a/.[12] described the pH-rate profile of the sulphite reductase activity of desulphoviridin from D. vulgaris and gave pH 5.5-6.0 as the region for optimum activity. The value found for the desulphoviridin from D. gigas is 7.0--7.5. Kobayashi and co-workers[12/ used a different assay method from the spectrophotometric one used here: their assay was done in Warburg vessels under a hydrogen atmosphere in the presence of hydrogenase and MV. Also, these workers found thiosulpbate and trithionate reductase activities for desulphoviridin which exhibited optimum pH's at 7.5 and 8.0 respectively. This is discussed in a later paper in this series. Desulphoviridin from D. gigas contains no flavins and cannot use flavins and/or NADPH as a reductant for sulphite reductase activity. Atomic absorption spectrophotometry indicates the presence of iron as the only metal in desulphoviridin from a group containing also Cu, Mn, Mo, Zn and Co. The concentration of iron is ca. 10 iron atoms per molecule of desulphoviridin. Extraction of the haem prosthetic group and its quantitation indicates 2 haems per molecule, and therefore suggests eight non-haem iron atoms. This was corroborated by a spectrophotometric method of non-haem iron analysis in view of the postulate[10/that desulphoviridin may not contain iron in the prosthetic group. Estimation of inorganic sulphide, S*, indicates ca. 8S*/molecule. It therefore appears that desulphoviridin contains 2 haems and 2 Fe4S4 centres per a2/32 molecule. E.P.R. evidence (25) also supports this view. If urea is removed from the desulphoviridin-urea incubation mixture by dialysis, the original electrophoretic behaviour is restored but activity is diminished: this loss of activity made it impossible to extract the a and /~ components and assay each for sulphite reductase activity. The optical absorption spectrum of desulphoviridin does not change when the enzyme is incubated either aerobically or anaerobically with sulphite and a variety of other ligands, thereby indicating that the enzyme does not react with its substrate in the oxidised state (unlike E. coli sulphite reductase (22) and the spinach nitrite reductase (23) which both form spectroscopically distinct complexes on incubation with various ligands). Reduction of the native desulphoviridin can be achieved by either dithionite or MVH, as shown by changes in the optical spectrum on addition of these reagents. Both reagents produce a decrease in absorbance at 450nm, seen most clearly in the difference spectrum; this is the area of the visible spectrum most commonly associated with the Fe/S centre [24]. Dithionite causes a decrease in the Soret band maximum but no shift, although reduction of the haem is indicated by the a-band shift from 580 to 550 nm. MVH, on the other hand, does shift the Soret band to higher wavelengths. Both reagents, therefore, can react with and presumably reduce the haem. In the presence of both dithionite and cyanide, desulphoviridin is further reduced, as is found for spinach nitrite reductase with these reagents[23]. Although the Soret band does not shift in this case, the a-band shifts from 580 to 550 rim. This further reduction is not seen with dithionite and nitrite. REFERENCES

I. A. B. Roy, and P. A. Trudinger, The Biochemistry of Inorganic Compounds of Sulphur. Cambridge UniversityPress (1970).

Iron-containing reductases 2. L. M. Siegel, H. Kamin, D. C. Rueger, R. P. Presswood, and Q. H. Gibson, Flavins and Flavoproteins (Edited by H. Kamin). University Park Press, Maryland (1971). 3. L. M. Siegel, P. S. Davis. and H. Kamin, .L Biol. Chem. 249, 1572 (1974). 4. M.J. Murphy, L. M. Siegel, H. Kamiu, D. V. Dervartaniau, J. P. Lee, J. LeGall, and H. D. Peck, Biochem. Biophys. Res. Comman. 54, 82 (1973). 5. M. J. Murphy, L. M. Siegel, H. Kamin and D. Rosenthal, J. Biol. Chem. 248, 2801 (1973). 6. A. R. Battersby, K. Jones, E. McDonald, J. A. Robinson, and H. R. Morris, Tet. Lett. 25, 2213 (1977). 7. J. Lee, J. LeGall, and H. D. Peck, J. Bact. 115, 529 (1973). 8. M. J. Murphy, and L. M. Siegel, J. Biol. Chem. 248, 6911 (1973). 9. G. W. Skyring, Proc. Aust. Biochem. 8, 21 (1975). 10. G. W. Skyring, and H. E. Jones, Austral. J. Biol. Sci. 30, 21 (1977). I1. K. Kobayashi, E. Takahashi and M. lshimoto, J. Biochem. 72, 879 (1972). 12. K. Kobayashi, Y. Seki, and M. Ishimoto, J. Biochem. 75, 519 (1974). 12. (a) J. R. Postgate, App. Microbiol. 11,265 (1963). (b) R. L. Starkey, Arch. Microbiol. 9, 268 (1938).

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(c) M. H. Hall, Ph.D. Thesis, Cambridge University, (1978). 13. H. E. Jones, and G. W. Skyring, Aust. J. Biol. Sci. 27, 7 (1974). 14. E. Layne, Meth. Enz. III, 447 (1957). 15. G. W. Skyring, and P. A. Trudinger, Can. J. Biochem. 19, 375 (1973). 16. K. Weber, and M. Osborn, J. Biol. Chem. 244.4406 (1969). 17. P. Andrews, Biochem. J, 96, 595 (1965). 18. K. Yagi, Methods of Biochemical Analysis (Edited by D. Glick), Vol. 10, p. 319, Interscience, New York (1962). 19. A. E. Harvey, J. A. Smart, and E. S. Amis, Anal. Chem. 27, 26 (1955). 20. N. Gilboa-Garber, Anal. Biochem. 43, 129 (1971). 21. J. P. Lee, and H. Peck, Biochem. Biophys. Res. Commun. 45, 583 (1971). 22. L. M. SiegeL, M. J. Murphy, and H. Kamin, J. Biol. Chem. 248, 251 (1973). 23. P. J. Aparicio, D. B. Knaff, and R. Malkin, Arch. Biochem. Biophys. 169, 102 (1975). 24. K. Kobayashi, S. Tachibana, and M. Ishimoto, J. Biochem. 65, 155 (1969). 25. M.H. Hall, R. H. Prince and R. Cammack, Biochem etBiophys. Acta, 581, 27 (1979).