Nitrobacter winogradskyi cytochrome b-559: a nonhaem iron-containing cytochrome related to bacterioferritin

Biochimica et BioptLvsicaActa, 976 (1989) 135-139 Elsevier

135

BBABIO 43050

Nitrobacter winogradskyi cytochrome b-559: a nonhaem iron-containing cytochrome related to bacterioferritin Toshihisa Kurokawa, Yoshihiro Fukumori and Tateo Yamanaka Department of Life Science, Faculty of Science. Tol~voInstitute of Technology. O-okavama, Meguro-ku. Tol~vo(Japan) (Received 31 January 1989) (Revised manuscript received 14 April 1989)

Key words: Cytochrome b" Nonheme iron; Bacterioferritin; (N. winogradskvi) A nonhaem iron-containing b-type cytochrome was purified to an electrophoretically h o m o g e n e o u s state from the soluble fraction of Nitrobacter winogradskyi, it showed absorption peaks at 532 and 418 nm in the oxidized form, and peaks at 559, 528 and 426 nm in the reduced form. The cytochrome has a native molecular weight of 260000. The molecular weight of the subunit was estimated to be 19500 by SDS-polyacrylamide gel electrophoresis analysis. O n e tool of protohaem and 8.6 g atoms of nonhaem iron were found in 39 kg of the cytochrome. N o inorganic sulphide was detected. Cytochrome b-559 was reduced by a flavoenzyme of N. winogradskyi with N A D P H in the presence of benzylviologen, and ferrocytochrome b-559 was easily oxidized by IV. winogradskyi ferricytochrome c-550. The combined properties of Nitrobacter cytochrome b-559, including the N-terminal sequence, are similar to those of Escherichia coli bacterioferritin.

Introduction C h a u d h r y et al. [1] found a b-type cytochrome in the soluble fraction of Nitrobacter winogradskyi and partially purified it. They studied a few of its properties. Although the b-type cytochrome does not seem to participate in nitrite oxidation by the bacterium [2], cytochrome b seems to be necessary for the reverse electron-transport system to produce N A D ( P ) H for reduction of CO 2 [3]. In the present studies, we have isolated cytochrome b-559 from the soluble fraction of the bacterium and determined some of its molecular properties. N. winogradskyi cytochrome b-559 is a nonhaem iron-containing cytochrome. Materials and Methods Organism Large-scale cultivation of Nitrobacter winogradslqvi ( A T C C 14123) was performed as previously described in 500 1 of inorganic medium using a stainless steel

Abbreviations: Mops, 4-morpholinepropanesulphonic acid; SDS, sodium dodecyl sulphate. Correspondence: T. Yamanaka, Department of Life Science, Faculty of Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152, Japan

fermenter of 600 1 capacity [4]. About 40 g of centrifugally packed cells were obtained from the 500-1 cultivation.

Physical and chemical measurements Spectrophotometric determinations were performed with a Shimadzu MPS-2000 multi-purpose spectrophotometer, using 1 cm light path cuvettes. Purity of the protein obtained was checked by polyacrylamide gel electrophoresis by the method of Davis [5]. Polyacrylamide gel electrophoresis in the presence of SDS was carried out according to Laemmli [6]. A m i n o acid composition was analysed with a Hitachi L-8500 a u t o m a t e d amino acid analyser after the sample had been hydrolysed with 6 M HCI for 24 h at 1 1 0 ° C in an evacuated sealed tube. The N-terminal amino acid sequence was determined by a gas-phase protein sequencer (Applied BioSystems Ltd., model 470A, U.S.A.). The content of iron in the cytochrome was determined by atomic absorption measurement with a Varian AA-875 atomic absorption spectrophotometer, after the protein preparation had been dialysed against 10 m M Tris-HCl buffer (pH 8.0) containing 10 m M E D T A . The content of inorganic sulphide was determined by the Methylene blue chromogen method of Fogo and Popowsky [7] as modified by Lovenberg et al. [8]. Protein content was determined by the method of Lowry et al. [9] with bovine serum albumin as the standard.

0005-2728/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

136

Special reagents N. winogradskyi cytochrome c-550 was purified as

cytochrome b-559 preparation.

previously described [4]. Mops was purchased from Sigma Chemical Co. (U.S.A.), DEAE-cellulose DE-32 from Whatman BioSystems Ltd. (England), and octylSepharose CL-4B and Sephacryl S-300 from Farmacia Fine Chemical (Sweeden).

Results

Purification of cytochrome b-559 Frozen cells (40 g in wet weight) of N. winogradskyi were suspended in 200 ml of 5 mM Tris-HC1 buffer (pH 8.0), and treated five times with a sonic oscillator (20 kHz, 250 W) for a total period of 50 rain. The suspension was centrifuged at 10000 x g for 20 min to remove unbroken cells. The resulting supernatant was further centrifuged at 186000 x g for 60 min to remove the membrane fractions. The supernatant obtained was fractionated with (NH4)2SO 4 and the precipitate which appeared between 40 and 60% saturation was collected by centrifugation at 30 000 × g and dissolved in 200 ml of 10 mM Tris-HCl buffer (pH 8.0). The solution obtained was dialysed against 10 mM Tris-HCl buffer (pH 8.0),' and applied to a DE-32 (DEAE-cellulose DE-32, Whatman) column (4 × 25 cm) which had been equilibrated with the same buffer as used for the dialysis. Cytochrome b-559 was eluted with a linear gradient produced from 400 ml each of 10 mM Tris-HCl buffer (pH 8.0) and 10 mM Tris-HCl buffer (pH 8.0) containing 0.3 M NaC1. Cytochrome b-559 in the eluates was monitored by the AalTnm/A4osn m ratio. The eluates which contained cytochrome b-559 were combined, dialysed against 10 mM Tris-HCl buffer (pH 8.0), and applied to the second DE-32 column (2.6 × 21 cm). After the column had been washed with 20 mM M o p s - N a O H buffer (pH 6.5), cytochrome b-559 was eluted with a linear gradient produced from 300 ml each of 20 m M Mops-NaOH buffer (pH 6.5) and 20 mM M o p s - N a O H buffer (pH 6.5) containing 0.2 M NaC1. A m m o n i u m sulphate was slowly added to the combined eluate which contained cytochrome b-559 to give a final concentration of 1 M, and the resulting solution was applied to an octyl-Sepharose CL-4B column (2 x 28 cm) which had been equilibrated with 50 m M Tris-HC1 buffer (pH 7.5) containing 1 M (NH4)2SO4. After the column had been washed with the same buffer as used for the equilibration, cytochrome b-559 was eluted with a linear gradient produced from 350 ml each of 50 m M Tris-HCl buffer (pH 7.5) containing 1 M (NH4)2SO 4 and 20 m M Tris-HCl buffer (pH 7.5). The eluted cytochrome b-559 fraction was concentrated by adsorption on and elution from a DE-32 column of an appropriate size and subjected to gel filtration on a Sephacryl S-300 column equilibrated with 50 m M Tris-HC1 buffer (pH 7.5) containing 0.25 M KC1. The eluates containing cytochrome b-559 were combined, concentrated by a DE-32 column as described above, and used as the purified

Spectral properties N. winogradskyi cytochrome b-559 showed absorption peaks at 418 and 532 nm in the oxidized form, and peaks at 559, 528 and 426 nm in the reduced form (Fig. 1). The cytochrome was not reduced with ascorbate or borohydride. The reduced form of the cytochrome in Fig. 1 was obtained by anaerobic addition of Na2S204. The reduction of the cytochrome proceeded slowly. In the presence of a small amount of benzylviologen its reduction rate by Na2S2Q 4 was much accelerated as previously reported for Azotobacter bacterioferritin [10-12]. Upon aeration, the reduced cytochrome was readily reoxidized to its original state. In contrast with the previous report [1], neither NO~ nor CO affected the spectrum of the reduced cytochrome. Therefore, the cytochrome did not seem to be cytochrome o. The pyridine ferrohaemochrome of the cytochrome showed absorption peaks at 419, 524 and 556 nm. This indicates that the cytochrome has protohaem as the prosthetic group. The millimolar extinction coefficient (~mM) per heme of the ct peak at 559 nm of ferrocyto-

08

0.6

U C 0

.1304

q6 !

/

z.18~ i -

I

-

Oxidized

. . . . .

Reduced

tO r-~

<

0.2 559

0

i

!

3OO

400

500

600

Wavelength (nm) Fig. 1. Absorption spectrum of N. winogradskyi cytochrome b-559. T h e cytochrome was dissolved in 50 m M Tris-HCl buffer (pH 7.5).

The cytochrome was anaerobically reduced by addition of a few crystals of Na2S~O4 to the oxidized cytochrorne in a Thunberg-type cuvette.

137 chrome b-559 was determined to be 24.8, assuming EmM at 557 nm of the pyridine f e r r o p r o t o h a e m o c h r o m e to be 34.4 [13]. A

w o

qln qlR

O

O

B

o

W

w

m

o

M

O

1

2

3

/-,

5

M

Fig. 2. SDS polyacrylamide gel electrophoresis of N. wmogradsl~vi cytochrome b-559 (lanes 1-3) and apo-cytochrome (lanes 4 and 5). The concentration of acrylamide was 16%. In lanes 2, 3 and 5 the preparation was treated at 1 0 0 ° C for 2 rain with 2% SDS in the presence of 570, 10 70 and 570 mercaptoethanol, respectively. In lanes 1 and 4, the preparation was treated at 1 0 0 ° C for 2 rain with 270 SDS in the absence of mercaptoethanol. M, marker proteins: from top to bottom, phosphorylase b ( M r = 93000), bovine serum albt..~min ( M r = 67000), ovalbumin ( M r = 43000), carbonic anbydrase (M~ = 29000) and horse cytochrome c ( M r = 12 500).

Molecular properties Fig. 2 shows the results of SDS-polyacrylamide gel electrophoresis of cytochrome b-559. In the gel made from 16% acrylamide, a single band was observed which corresponded to the protein species of 19.5 k D a when the cytochrome was heated at 1 0 0 ° C in the presence of SDS and fl-mercaptoethanol before being subjected to the electrophoresis, or when a p o c y t o c h r o m e m a d e by treating the cytochrome with HCl-acetone was subjected to the electrophoresis. When the cytochrome was boiled at 1 0 0 ° C with SDS in the absence of fl-mercaptoethanol before being subjected to the electrophoresis, two bands were observed which corresponded to the protein species of 17 and 19.5 kDa, respectively. In all cases, nonhaem iron was not detected on the gel staining with thioglycolic acid and bathophenanthroline disulphonate [14]. Table I shows the amino acid composition of cytochrome b-559 together with those of other bacterioferritin-like b-type cytochromes. The a m o u n t s of Asx + Glx and I1e + Leu of the cytochrome were as large as found for other bacterioferritin-like b-type cytochromes. The molecular weight of the apoprotein was calculated to be 37 800 per protoheme from the amino acid composition. It was also estimated to be 38000 on the basis of the

TABLE I

Comparison of amino acid composition of N. winogradvkyi cvtochrome b-559 with those of several bacterioferritin-like b-type cvtochromes n.d., not determined. Amino acid

Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe Lys His Arg Cys Trp Mr

Nitrobacter winogradsk:vi

A zotobacter oinelandii

A zotobacter chroococcum

Escherichia coil

Rhodobacter sphaeroides

(present study)

[12]

[17]

[15]

[191

21 5 5 21 3 8 13 6 6 13 18 7 4 14 9 6 n.d. 3 18 900

17.2 2.2 5.3 26.8 2.3 9.9 8.0 1.9 4.4 12.2 25.7 5.7 2.1 11.7 6.9 3.1 2.3 2.4 17 000

16.3 7.9 7.5 19.8 3.1 13.4 14.2 10.4 3.4 7.6 19.1 3.9 5.8 8.7 3.6 5.4 n.d. n.d. 17 000

24 2 4 25 1 11 8 6 7 11 24 7 3 9 5 9 0 2

19 5 9 20 3 10 18 4 4 8 20 6 4 8 7 8 3 1

18 495

17 300

138

I

I0

20

30

40

50

N. winogradskyi cytochrome b-559

D

E. coli

GN EL~ V

W

Y

iii

AKKWRA ~-I

E D

bacterioferritin

Fig. 3. Comparison of N-terminal amino acid sequence of N. winogradskyi cytochrome b-559 with that of E. cob bacterioferritin [15]. The boxed residues are identical between the two proteins.

haem and protein contents measured by the Lowry assay (26.3 nmol per mg protein). The cytochrome contained 9.6 iron atoms per protoheme molecule when the metal content was determined by atomic absorption measurement, while it did not have inorganic sulphide when the sulphide content was measured by the methylene blue chromogen method [7,8]. When the cytochrome was subjected to the gel filtration with a Sephacryl S-300 column, a single peak was observed in the elution profile. The molecular weight of the protein contained in the peak was estimated to be 260 000 (data not shown). Therefore, N. winogradskyi cytochrome b559 seemed to exist in a polymeric form in aqueous solution. The N-terminal sequence of the first 50 residues was determined by a gas-phase protein sequencer. When the sequence was compared with that of E. coli bacterioferritin [15], 30 residues were identical (Fig. 3).

with benzylviologen radical. Ferrocytochrome b-559 reduced rapidly N. winogradsl£vi ferricytochrome c-550. Discussion

Cytochrome b-559 purified from N. winogradskyi in the present study seems to be identical to the b-type cytochrome described by Chaudhry et al. [1]. However, ferrocytochrome b-559 does not react with N O { or CO unlike their results. Therefore, although cytochrome b559 is highly auto-oxidizable, it does not seem to be cytochrome o. One of the most interesting properties of cytochrome b-559 is that it contains nonhaem iron although it has no inorganic sulphide. In this respect, N. winogradsk.vi cytochrome b-559 may be classified among bacterioferritins. Thus, the N-terminal sequence of the N. winogradskyi cytochrome resembles that of E. coli bacterioferritin [15]. The molecular weight of the subunit, the haem content, and the large native molecular weight of N. winogradskyi cytochrome b-559 are comparable to the respective features of bacterioferritins [12,13,17,18] (Table II). Cytochrome b-559 is similar to E. coli, A. vinelandii and A. chroococcum bacterioferritins also in high contents of Leu and Ile, although its amino acid composition is fairly different from those of the bacterioferritins [12,17,18]. Similar proteins have been isolated from the non-sulphur purple bacteria. Cytochrome b-558 from Rhodobacter sphaeroides [19] and cytochrome b-557.5 from Rhodospirillum rubrum

Reduction by NADPH-cytochrome c reductase As has been mentioned, cytochrome b-559 was not reduced by ascorbate or borohydride. Therefore, its Era.7 value seemed to be zero volt or lower. In the presence of benzylviologen, N. winogradskyi N A D P H cytochrome c reductase [16] catalysed anaerobic reduction of cytochrome b-559 with N A D P H as the electron donor. This reaction was dependent on the amount of benzylviologen added. In the absence of benzylvioiogen, the cytochrome was not reduced at all by the enzyme with N A D P H . N A D P ÷ was not reduced by the enzyme

TABLE II

Comparison of N. winogradslqvi cytochrome b-559 with several bacterioferritin-like b-type vytochlomes Properties

a peak (nm) M r of cytochrome in solution M, of subunit M, per haem Content of nonhaem iron per haem

N. winogradskyi

A. vinelandsii

A. chroococcum

E. coil

Rb. sphaeroides

R. rubrum

Cyt b-559

Cyt b-557.5

Cyt b-557.5

Cyt b-557

[121

[17]

[15,]81

Cyt b-558 [191

[201

559

557.5

260000

660000

19500 39 000

17000 37 000

8.6

100

557.5 n.d. 17000 n.d. n.d.

n.d., not determined; Cyt, cytochrome. " Calculated on the basis of the data in Ref. 19 assuming M r per haem to be 30000.

557 269000 or 452 000 15000 n.d. 67 "

Cyt b-557.5

558

557.5

> 100 000

450000

17000 -- 34000

23 000 n.d.

n.d.

n.d.

139 [20] are very similar to N. winogradskyi c y t o c h r o m e b-559 in m a n y properties. A l t h o u g h the iron c o n t e n t of the c y t o c h r o m e s b from the p h o t o s y n t h e t i c bacteria has not been d e t e r m i n e d , Rb. sphaeroides c y t o c h r o m e b-558 appears to be related to b a c t e r i o f e r r i t i n on the basis of native size, s u b u n i t s t r u c t u r e a n d h a e m c o n t e n t . A m o n g the p r o t e i n s related to bacterioferritins, the a m i n o acid c o m p o s i t i o n of Rb. sphaeroides c y t o c h r o m e b-558 seems to be most similar to the c o m p o s i t i o n of N. winogradsk),i c y t o c h r o m e b-559. T h e similarity in the a m i n o acid c o m p o s i t i o n b e t w e e n these two c y t o c h r o m e s b seems not s u r p r i s i n g b e c a u s e N. winogradslg, i is p h y l o g e n e t i cally close to n o n - s u l p h u r p u r p l e bacteria as j u d g e d from the p r i m a r y s t r u c t u r e of c y t o c h r o m e c [21] a n d the base s e q u e n c e of o l i g o n u c l e o t i d e s of 16 S r i b o s o m a l R N A [221. T h e results of SDS-gel e l e c t r o p h o r e s i s of N. winogradskyi c y t o c h r o m e b-559 seem i n t e r e s t i n g ; the p r o t e i n gives two b a n d s c o r r e s p o n d i n g to 17 a n d 19.5 k D a p r o t e i n species when subjected to electrophoresis w i t h o u t ]3-mercaptoethanol. As the a p o c y t o c h r o m e gives o n e b a n d c o r r e s p o n d i n g to 19.5 k D a p r o t e i n species, the 17 k D a p r o t e i n species m a y have kept p r o t o h a e m for a m o m e n t after the electrophoresis has started. Thus, the c y t o c h r o m e has o n e p r o t o h a e m molecule per two s u b u n i t s . However, it is n o t u n d e r s t o o d clearly at p r e s e n t why the s u b u n i t with the h a e m migrates m o r e rapidly. T h e physiological f u n c t i o n of the b a c t e r i o f e r r i t i n is still unclear. The p r o t e i n is p o s t u l a t e d to act as an i r o n - s t o r a g e p r o t e i n or as an e l e c t r o n - s t o r a g e protein. As N. winogradskyi c y t o c h r o m e b-559 is easily reduced by N A D P H - c y t o c h r o m e c reductase of the b a c t e r i u m in the presence of b e n z y l v i o l o g e n a n d f e r r o c y t o c h r o m e b-559 readily reduces f e r r i c y t o c h r o m e c-550 of the b a c t e r i u m , it seems likely that c y t o c h r o m e b-559 acts as o n e of the electron carriers in the reverse e l e c t r o n - t r a n s port system from c y t o c h r o m e c-552 to N A D P ÷ [2]. T h e n o n h a e m iron a t o m s in c y t o c h r o m e b-559 m a y act as an electron reservoir.

Acknowledgements T h e a u t h o r s wish to t h a n k Drs. K. Ikeda a n d S. I n o u e ( D e p a r t m e n t of Biochemistry, O s a k a U n i v e r s i t y

of P h a r m a c e u t i c a l Sciences, Osaka, J a p a n ) for their kind help in a n a l y s i n g a m i n o acid c o m p o s i t i o n of cytoc h r o m e b-559, a n d Dr. Y. T a m a u r a ( T o k y o I n s t i t u t e of T e c h n o l o g y , T o k y o , J a p a n ) for his kind help in det e r m i n i n g iron c o n t e n t .

References 1 Chaudhry, G.R., Lees. H. and Suzuki. I. (1980) Can. J. Microbiol. 26, 850-851. 2 Tanaka, Y., Fukumori, Y. and Yamanaka, T. (1983) Arch. Microbiol. 135, 265-271. 3 Aleem. M.I.H. and Sewell. D.L. (1984) in Microbial Chemoautotrophy (Strohl, W.R. and Tuovinen, O.H., eds.), pp. 185-210, Ohio State University Press, Columbus, OH. 4 Yamanaka, T.. Tanaka, Y. and Fukumori, Y. (1982) Plant Cell Physiol. 23, 441-449, 5 Davis, B.J. (1964) Ann. NY Acad. Sci. USA 121,404-427. 6 Laemmli, U.K. (1970) Nature 227, 680-685, 7 Fogo. J.K. and Popowsky, M. (1949) Anal. Chem. 21. 732-735. 8 Lovenberg. W.. Buchanan. B.B. and Rabinowitz, J.C. (1963) J. Biol. Chem. 238, 3899-3913. 9 Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 10 Bulen, W.A., LeConte, J.R. and Lough, S. (1973) Biochem. Biophys. Res. Commun. 54, 1274-1281. II Stiefel, E.I. and Watt, G.D. (1979) Nature 27'9, 81-83. 12 Li, J., Wang, J., Zhong, Z., Tu. Y. and Dong. B. (1980) Sci. Sinica 23. 897-904. 13 Falk, J.E. (1964) Porphyrins and Metalloprophyrins, p. 240, Elsevier, Amsterdam. 14 Oomori, D,, Tanaka, Y. and Suzuki, K. (1981) Seikagaku 53. 703. 15 Andrews, S.C., Smith, J.M.A., Guest, J.R. and Harrison, P.M. (1989) Biochem. Biophys. Res. Commun. 158, 489-496. 16 Kurokawa, T. Fukumori, Y. and Yamanaka, T, (1987) Arch, Microbiol. 148, 95-99. 17 Chen, M. and Crichton, R.R. (1982) Biochim. Biophys. Acta 707, 1-6 18 Yariv, J., Kalb. J., Sperling, R., Bauminger. E.R., Cohen. S.G. and Ofer. S. (1981) Biochem. J. 197. 171-175. 19 Meyer, T.E. and Cusanovich, M.A. (1985) Biochim. Biophys. Acta 807, 308-319. 20 Bartsch, R.G.. Kakuno. T., Horio, T. and Kamen, M.D. (1971) J. Biol. Chem. 246, 4489-4496. 21 Tanaka, Y., Fukumori, Y. and Yamanaka, T. (1982) Biochim. Biophys. Acta 707, 14-20. 22 Seewaldt, E., Schleifer, K.-H,, Bock. E. and Stackebrandt, E. (1982) Arch. Microbiol. 131. 287-290.