Biochimica et Biophysica Acta, 322 (1973) 133-14o © Elsevier Scientific Pub l i sh ing C ompany , A m s t e r d a m - P r in ted in The Ne the r l ands

BBA 36501

FERREDOXIN FROM V E I L L O N E L L A A L C A L E S C E N S

H O W A R D D A L T O N AND J O N Z U B I E T A

School of Molecular Sciences, University of Sussex, Brighton, BNI 9QJ (Great Britain)

(Received F e b r u a r y I9th , 1973)

SUMMARY

Ferredoxin from Veillonella alcaLescens was isolated and purified giving an Aa9o/A~8 o ratio of 0.82. Amino acid assays, iron and acid-labile sulfide analysis and E PR measurements indicate that the protein belongs to the class of eight iron-eight acid-labile sulfide ferredoxins. The redox potential, E 0 ---- --41o mV, was measured by polarographic techniques.

INTRODUCTION

Ferredoxin from Micrococcus lactilyticus, classified as Veillonella alcalescens by Rogosa 1, was previously isolated by Valentine et al. 2 who demonstrated that it mediated hydrogen evolution from hypoxanthine. The reaction was not specific for the host ferredoxin, since ferredoxin from Clostridium pasteurianum could be substi- tuted in the reaction. Ferrodoxin from V. alcalescens acted as an electron acceptor from xanthine dehydrogenase, being more effective than artificial electron acceptors ~. In the course of study of xanthine dehydrogenase from V. alcalescens it was necessary to purify the ferredoxin for kinetic observations. We report here the purification and some properties of ferrodoxin from this organism.

MATERIALS AND METHODS

Chemicals Sephadex G-5o, Sephadex G-5o Superfine and Blue dextran 2ooo were obtained

from Pharmacia Fine Chemicals. Cytochrome c (horse heart, mol. wt I I 7oo), myo- globin (tool. wt 17 8oo), chymotrypsin (tool. wt 22 5oo), ovalbumin (tool. wt 43 ooo), beef pancreas deoxyribonuclease and protamine sulfate grade II were obtained from Sigma Chemical Co. Whatman DE 32 DEAE-cellulose was obtained from W and R Balston Ltd. Ferredoxin from C. pasteurianum (tool. wt 6000) was obtained from Worthington Biochemicals.

All other chemicals used were the best purity commercially available.

134 H. DALTON, J. ZUBIETA

Growth of Veillonella alcalescens The organism, a gift from Dr P. N. Hobson, Rowett Research Institute, Aber-

deen, was grown on a modified medium of Whitely and Douglas 4. The amounts of medium components were substantially different from those originally used and will be given in detail elsewhere (Dalton, H., unpublished). The organism was grown anaerobically for 8 h at 37 °C in 4oo-1 all-glass fermentors.

Iron and acid-labile sulfide analyses Iron was determined colorimetrically ~ and by atomic absorption spectroscopy.

Acid-labile sulfide was determined by the method of Gilboa-Garber 15.

Disc gel eleetrophoresis Gels were prepared according to the method of Hendrick and Smith 6 to give

final acrylamide concentrations of 7.5%, 15% and 3O9/o at pH 8.5 and 7.5% and 15% at pH 4.5- Duplicate gels were loaded with I5-/zg and 5o-#g amounts of protein.

Molecular weight determination Thin-layer plates of Sephadex G-5o Superfine were prepared as described in the

Pharmacia handbook and run at room temperature in the thin-layer apparatus with appropriate standards.

Amino acid analysis I-mg samples of pure ferredoxin in glass ampoules were hydrolysed in 6 M HC1

at IiO °C for 16, 24, and 72 h. The hydrolysates were evaporated to dryness in about 20 rain, stored under vacuum over P~O 5 and then analysed on a Locarte amino acid analyzer. Performic acid oxidation was performed according to the method of Moore 7.

Purification of ferredoxin Approximately 200 g wet weight of bacteria were suspended in i 1 of o.05 M

potassium phosphate buffer pH 7.9 and disrupted in the French pressure cell at 2 tons/inch 2. The extrudate was again subjected to a similar treatment, i mg DNAase was then added to the viscous extract, allowed to stand at 4 °C for 30 rain, and then centrifuged at 23 ooo × g for 30 rain at 4 °C. The brown supernatant was then treated with 3% protamine sulfate and the precipitate removed by centrifugation at 23 ooo × g for 20 rain. The supernatant was applied to column of DE 32 (ii cm X 8 cm dia- meter), and the column washed successively with approximately I 1 0.05 M, 500 ml o.I M and I 1 0.2 M phosphate buffer, pH 7.8. The dark brown band of ferredoxin at the top of the column was slowly etuted by tile 0.2 M buffer and was preceded by a red band of rubredoxin. The ferredoxin was eventually eluted with o.25 M buffer.

The fractions containing ferredoxin were diluted 5-fold and loaded on to a second DE column (2o cm × 3 cm diameter). After washing the column with 5 bed volumes of o.o 5 M phosphate buffer, contaminating proteins were removed by suc- cessive additions of o.I, o.15 and o.175 M buffer. Ferredoxin was finally removed with 0.2 M buffer. The ferredoxin was then concentrated by blowing nitrogen gas over the surface overnight and applied to a column (5o cm × 3 cm) of Sephadex G-5o previ- ously equilibrated in 0.02 M phosphate buffer, pH 7.5- Ferredoxin was eluted with tile same buffer, and stored by the method of choice given in Results.

FERREDOXlN FROM Veillonella alcalescens 135

RESULTS

Purity Ferredoxin Furity was checked by four independent methods: (I) Disc gel

electrophoresis which gave a single band on gels at pH values of 4-5 and 8.5 at the acrylamide concentrations given in Methods. (2) Aago/A2so ratio. Ferredoxin was purified to a constant A~9o/A~8 o ratio of 0.82. This value is comparable to all other bacterial ferredoxins which have been purified 1°. (3) EPR. Purified ferredoxin samples (IO mg/ml) were checked for the presence of a g = 4.3 signal in the oxidized state. Absence of such a signal was indicative of the absence of rubredoxin from the sample. (4) Amino acid analysis. Samples were checked for the presence of an unchar- acteristically high number of any amino acid.

Absorption spectrum The spectrum of purified ferredoxin as isolated is shown in Fig. i . Addition of

excess sodium dithionite resulted in complete loss of the shoulder at 31o nm and the peak at 39 ° n m .

Stability After elution of ferredoxin from the Sephadex G-5 o column the A3,o/A28 o ratio

was 0.82. Table I gives the effect on this ratio of different storage methods. Material which had been either frozen at --30 °C, or freeze dried kept in liquid

nitrogen for two weeks was rechromatographed on DE 32 cellulose. Elution of the ferredoxin with 0.25 M phosphate buffer gave fractions in which the A39o/A28 o ratio was 0.78. These fractions were applied to and eluted from Sephadex G-5o to give fractions with a ratio of 0.82. Clearly, then, the low ratio material contained a pro- portion of material with a normal Aa,o/A28 o ratio of 0.82. Consequently, ferredoxin was kept in liquid nitrogen and rechromatographed before use.

0.4

0.2

wavelength nm

Fig. I. Ul t ravio le t -v is ib le s p e c t r u m of oxidized fer redoxin f rom V. alcalescens.

136 H. DALTON, J. ZUBIETA

T A B L E I

Method of storage Days A a9o/.4 2,0

Freeze dr ied f rom NH4HCO a in o.I M p h o s p h a t e buffer, p H 7.5, and k e p t under N 2 a t - -3 ° °C 14

Frozen a t --3 ° °C 14 Frozen a t - -3 ° °C under N~ 14 o °C under N 2 14 Pel le ted and kep t in l iqu id n i t rogen 14

o.71 0.65 0.68 o.71 0.75

Molecular weight The molecular weight of the ferredoxin was estimated by comparison with

known molecular weight proteins on thin-layer Sephadex plates (see Methods). Fig. 2 is a plot of the distance travelled by the protein against the log of each of their respec- tive molecular weights. Ferredoxin from Veillonella was estimated to be 6000 daltons.

Molar extinction coefficient The absorbance of a solution of ferredoxin in NH4HCO a buffer was measured

at 39 ° n m by dilution of an aliquot into 0.05 M phosphate buffer, pH 7.8. 2.5 ml of undiluted solution were placed in a pre-weighed weighing bottle. The bottle, along with 3 others containing 2.5 ml of NH4HCO 3 were placed in a desiccator containing P20~ and dried at 4 ° °C under vacuum s. Before weighing, the samples were removed and cooled to room temperature in a desiccator. The sample and blanks reached a constant weight after about 64 h of drying. An average value for the Ea90 was 29 60o ± 400.

Iron content Iron determination gave a value of 8.2 ~ o. 4 atoms per molecule of 6ooo dal-

tons by the colorimetric technique (see Methods) and 8. 7 ~- 0.3 atoms per molecule by atomic absorption spectroscopy.

Acid-labile sulfide Acid labile sulfide was determined as 7.6 ± o.6 atoms per molecule of 6ooo

daltons in two separate determinations.

Amino acid content Results of amino acid analysis are shown in Table II and compared with the

assays for Micrococcus aerogenes and C. acidi-urici ferredoxins.

Titration of V. alcalescens ferredoxin with sodium dithionite Previous workers 9 have demonstrated by EPR spectroscopy that clostridial

ferredoxins accept two electrons from sodium dithionite for complete reduction. Ad- dition of the first electron produces a nearly axial signal upon which is superimposed a rhombic signal on addition of the second electron.

The effect of increasing dithionite concentration on the EPR spectrum of ferredoxin (Asgo/A2so = 0.82) was tested. Addition of two electrons gave a reduced

~ERREDOXlN FROM Veillonella alcalescens 137

T A B L E I I

DETERMINATION OF MOLAR EXTINCTION COEFFICIENT OF FERREDOXIN FROM reillonella alcalescens The sample used has an absorbance of 12.o at 39o nm and was prepared and dried as described in the text.

Drying time ]Vet weight Calculated amount (h) (mg) of ferredoxin

( t*moles /ml )

~'390 nm

4 ° 6.56 o.419 28 600 64 6.34 0.405 29 600 88 6.41 o.4o9 29 3oo

lO2 6.32 0.4o4 29 700

spectrum which did not change upon further addition of electrons (see Fig. 2). Further investigations on the EPR species involved is in progress.

Measurement of oxidation-reduction potential A recent report 1~ used a polarographic technique to determine the redox poten-

tial of ferredoxin from C. pasteurianum. A value of --570 mV was found for the E0 at pH 7.0 which varied by only lO% between pH values of 6.5 and 9.0. The E0 value generally found for ferredoxins using the methyl viologen dye method is around --42o inV.

Ferredoxin from V. alcalescens was found to have an E 0 value of --41o mV at pH 7.6. A plot of log E(ia-i)/iJ against potential yielded a straight line with slope 0.029. This indicates that the redox process is reversible and involves approximately two electrons. Further polarographic studies on related iron-sulfur proteins are cur- rently in progress.

?

E

3(

2( mY°gl°binu~rn°t rY P sin

c ,oc ro ~-~rubredoxin (D.be rre)

v 0,c.!, / 'Ierredoxin (CL past.)

i I I I 200 250 300

distance migrated mm Fig. 2. Molecular weight determinat ion for ferredoxin from V. alealescens, plot indicating distance migrated on Sephadex G-5o Superfine plate versus molecular weight.

138 H. DALTON, J. ZUBIETA

T A B L E l I I

A M I N O A C I D C O M P O S I T I O N O F V. alealescens F E R R E D O X I N

Amino acid Nearest integer Nearest integer Average M. C. from hydrolysis" from oxidation** aerogenes§ acidi-urici§

Asx 5 5 5 8 9 T h r ' * * 2 2 2 o I

Ser*** 7 7 7 5 3 ( ; I x 5 6 6 4 4 Pro 2 2 2 5 4

G l y 5 4 5 4 4 Ala 5 4 5 7 9 V a l t 3 3 3 4 6 M e t t - i - -

l i e ? 7 5 6 6 5 L e u . . . . . . T y r 2 2 2 2

P h e . . . . . H i s . . . . . L y s 2 2 2 I - A r g I I I - I C y s ( O a H ) - 8 8 8 8 T r p t t . . . .

55 54 55 Total

* The results are the average of three values determined at 16, 24 a n d 72 h. ** The performic acid ox idat ion results are the average of three values d e t e r m i n e d at 16,

48 a n d 72 h . *** I n determining the best ratio for serine and threonine, the results were extrapolated to

zero t ime. ? The best ratio for val ine and isoleucine is dependent on the 72-h recoveries.

*t T r y p t o p h a u was determined by colorimetric analys is by the meth o d of Opienska-B lauth el a/, 14.

§ See Tanaka el al. ag, for amino acid compos i t ions of these and other e ight i ron-e ight sulfide ferredoxins.

Physiological role of Veillonella ferredoxin A crude extract of the organism was passed through a small DEAE-cellulose

column to remove ferredoxin 2. The extract thus prepared would not evolve hydrogen when either pyruvate, a-ketoglutarate, xanthine or hypoxanthine was added, but did so on addition of purified ferredoxin. Earlier workers x° had observed this using ferre- doxin of doubtful purity. Utilizing a purified sample (Aago/A~8o = o.82), the activity of the ferredoxin was estimated at 21 units/rag as determined in the phosphoroclastic assay procedure according to Mortenson et al. n.

DISCUSSION

The iron and labile-sulfide analyses, together with the amino acid assay which demonstrates the presence of eight cysteine and two aromatic residues 16, indicate that ferredoxin from V. alcalescens is a member of the class of eight iron-eight acid-labile sulfide proteins for which an X-ray crystallographic structure is now available for the cases of M. aerogenes ferredoxin 17,~8. The EPR and polarographic studies place an upper limit of two on the number of electrons accepted per molecule of ferredoxin. The EPR-monitored dithionite titration indicates the presence of two distinct para-

FERREDOXIN FROM Veillonella alcalescens 139

2 9 0 0 i i 3,00 33'00 35'oo 3700

Fig. 3. E P R s p e c t r u m of reduced fer redoxin f rom V. alcalescens. Aliquots (0.2 ml) con ta in ing 0 .52/~mole /ml fe r redoxin in 0.05 M p o t a s s i u m p h o s p h a t e buffer, p H 7.8, were m a d e anaerobic in evacuab le E .P .R . t ubes by r epea ted evacua t i on and gass ing wi th a rgon con ta in ing 2% hydrogen . To each t u b e an anaerobic solut ion of 42.80 m M sod ium di th ioni te in 0.05 M p o t a s s i u m p h o s p h a t e buffer, p H 7.8, was added to give up to two electrons per molecule of ferrodoxin. The so lu t ion of sod ium di th ioni te was s t anda rd i zed i m m e d i a t e l y before and af ter use aga ins t s t an - dard p o t a s s i u m ferr icyanide wi th m e t h y l viologen as indicator . After mix ing t he d i th ioni te wi th the fer redoxin for I m i n t he samples were frozen in l iquid n i t rogen and t he E P R spec t ra ob- t a ined us ing a Var i an E9 E P R spec t romete r wi th t e m p e r a t u r e control as described by Lowe et al. 13. The E P R condi t ions were as follows : f r equency ioo kHz, mic rowave power IO nlW, scan t ime 2 min , t ime c o n s t a n t o. i s, t e m p e r a t u r e 21 °K, modu l a t i on amp l i t ude io G, opera t ing f r equency 9.143 GHz, field in G as indicated.

magnetic sites. This observation is in accord with the previous investigations on bacterial eight-iron ferredoxins carried out by Orme-Johnson and Beinert 9 which suggested the presence of two iron-sulfido "cubane" clusters as found in M. aerogenes ferredoxin 18.

A striking feature of the amino acid composition is the presence of a methionine residue, an amino acid not found in the eight iron ferredoxins from Clostridium and Micrococcus 19, although methionine is present in the pr imary structure of the four- iron ferredoxin from Desulfovibrio gigas 2°. V. alcalescens ferredoxin is also seen to contain an unusually large number of basic amino acid residues 21 (2 lysine, I arginine) when compared with the similar ferredoxins from M. aerogenes and C. acidi-urici.

ACKNOWLEDGEMENTS

We thank Mr D. J. Lowe for measuring the E P R spectra, Mr D. Watson for the amino acid analysis, Dr C. M. Elson for the polarographic measurements and Dr R. C. Bray and Professor J. R. Postgate for providing facilities. H.D. wishes to thank the Medical Research Council for financial support (to R.C.B.), and J.A.Z. expresses his gratitude to the National Institutes of Health, U.S.A., for a postdoctoral research fellowship.

R E F E R E N C E S

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453-456

14o H. DALTON, J. ZUBIETA

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