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Purification and properties of two ferredoxins from the nitrogen-fixing bacterium Bacillus polymyxa
ARCHIVES
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
and
633-640 (1973)
168,
Properties
Nitrogen-Fixing
of Two
Bacterium DUANE
Department
of Cell Physiology,
Ferredoxins
Bacillus
from
the
polymyxa'
C. YOCH
University
of California,
Received
Berkeley,
California
94720
May 3, 1973
Two ferredoxins, designated FdI and FdII, have been isolated from the nitrogenfixing bacterium Bacillus polymyza. The two ferredoxins were readily separated on DEAE-cellulose and disc gel electrophoresis. The amino acid compositions of FdI and FdII showed them to be different protein species; the greater number of acidic amino acid residues in FdI than in FdII appears to account for separation based on electronic charge. FdI and FdII were both found to have four nonheme iron and folu acid-labile sulfur groups per mole. The absorption spectra of the two ferredoxins are almost identical, with a peak in the visible region of the spectrum at 385 nm. The Aass/A~so absorbance ratio of both ferredoxins was 0.540.55. FdII was not stable under aerobic conditions, as indicated by a decrease in the visible region of the spectrum. Both FdI and FdII have nearly identical molecular weights, as judged by gel filtration and amino acid composition (approx 8800). The purified ferredoxins catalyzed the photoreduction of NADP by spinach chloroplasts with equal effectiveness. In the nitrogen-fixation reaction of B. polymyxcz, FdII was more effect.ive than FdI.
Recent studies in this laboratory on bacterial ferredoxins have shown that several species of bacteria have not one but two biologically active ferredoxins. Rhodospirillum rubrum, a facultative phototroph, was the first organism from which two types of ferredoxin were isolated (1, 2). These ferredoxins, referred to as types I and II (FdI and FdII), differed in iron-sulfur content, amino acid composition, spectra, and molecular weight. This was the first demonstration that ferredoxins, like cytochromes, can occur in the cell in several forms. A second organism from which two biologically active ferredoxins were isolated is the nitrogen-fixing obligate aerobe Axotobatter vinelandii (3). The two ferredoxins (designated azotobacter FdI and FdII) differed in spectral properties, oxidationreduction potential, and size. Both ferrer Part of this work was presented in a paper given at the Dinitrogen (NJ Fixation Seminar at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, May 9, 1973. 633 2opyright Ul rights
@ 1973 by Academic Preen, of reproduction in any form
Inc. reserved.
doxins from Azotobacter functioned in the nitrogenase reaction of that organism and were able to replace spinach ferredoxin in the photochemical reduction of NADP by spinach chloroplast fragments (3, 4). In the course of an investigation aimed at characterizing a ferredoxin (5) from the nitrogen-fixing bacterium Bacillus polymyxa, Yoch and Valentine (6) made the preliminary observation that this organism, like R. rubrum and A. vinelandii, contained tlvo ferredoxins. These ferredoxins were distinguished from one another by their elution characteristics on DEAE-cellulose; tn-o biologically active ferredoxin fractions were obtained. Further purification of the recombined ferredoxin fractions resulted in the isolation of only one species of (near homogeneous) ferredoxin. It had the unusual property of containing four atoms of nonhcme iron and acid-labile sulfur per molecule (6, 7) making it an intermediate form compared to the eight iron-sulfur bacterial ferredoxins and the two iron-sulfur plant ferredoxins. The fate of the second ferredoxin was a mat,-
634
YOCH
ter of speculation
in the preliminary report (6). This paper presents evidence that two chemically distinct species of ferredoxin (designated FdI and FdII) exist in B. po&myza. FdI corresponds to the protein initially described by Shethna et al. (5) and later characterized by Orme-Johnson et al. (7) and Yoch and Valentine (6) ; FdII is the protein initially reported to be lost during the purification process (6). These ferredoxins differ in amino acid composition, biological activity in the B. polymyxa nitrogenase reaction, and stability in air (FdII is oxygen-labile). They appear to be identical in iron-sulfur content, molecular weight, and spectral characteristics. METHODS Culture. Bacillus polymyxa strain Hino (obtained from Dr. R. V. Klucas of the University of Nebraska) was grown in 12-liter carboys in medium containing (g/liter) : K~HP04.3Ht0, 14.3; KH2POI, 11.8; CaC12.2Ht0, 0.05; MgSOd, 0.25; N&l, 0.01; FeS04.7Hz0, 0.015; Na2Mo04. 2H20, 0.005; biotin, 0.00005; sucrose, 20; and (NH4)*S04, 2.0, as nitrogen source. NO precautions were taken to make the cultures anaerobic other than filling the carboys to the neck and covering them with aluminum foil. After 24-36 hr (29-31”C), the cultures were harvested and used for the isolation of ferredoxin. Nitrogen-fixing cultures were obtained by omitting the (NH4)$04 from the medium and sparging the culture with Nz gas. Isolation of jerredoxin. Cells (1500 g wet wt) grown on the (NH4)2S04 medium were suspended in 3 vol of 0.02 M phosphate buffer, pH 7.4, and disrupted by sonication for 5 min at 5°C with a 20.kHz Branson sonifier at full power. The disrupted cells and debris were incubated with 2 mg of deoxyribonuclease at room temperature for 1 hr and then centrifuged at 32,OOOgfor 10 min. The resulting supernatant. fluid was passed over a bed of DEAE-cellulose (6 X 20 cm) equilibrated with 0.02 M phosphate buffer, pH 7.4. This buffer was used throughout the purificat.ion process. The ferredoxin was adsorbed at the top of the column as a brown band. The column was washed with loo-ml portions of buffer containing no salt, 0.1 M NaCl, and 0.2 M NaCl. The ferredoxin was eluted from the column with buffer containing 0.4 M NaCl; the ferredoxin solution was then dilut,ed fivefold and charged on a DEAE-cellulose chromatography column (1.5 X 35 cm). Discontinuous elution of the column with 75.ml portions of 0.28, 0.30, and 0.35 M NaCl in phosphate buffer
showed two distinct ferredoxin bands eluting from the column. From this point on, the two ferredoxins were each purified separately, although identical procedures were used. FdII was stored under argon between purification steps. The ferredoxins (buffered with 0.1 M phosphate buffer, pH 7.4) were further purified by ammonium sulfate fractionat,ion. The colorless precipitates that formed between 0 and 65% ammonium sulfate saturation were removed by centrifugation (35,000g for 10 min) and discarded. The ferredoxins were then precipitated by the further addition of ammonium sulfate to 100% saturation, the pellets were dissolved in 2.0 ml of buffer and were individually chromatographed on a Sephadex G-100 column (1.5 X 85 cm). Fractions with absorbance ratios A&A280 between 0.54 and 0.55 were pooled and used for characterization of the two ferredoxins . Although the ferredoxins isolated, purified, and characterized in this study were from NH,+grown cells, the same two ferredoxins were also isolated from Nz-grown cultures of B. polymyza. Analytical procedures. The molecular weights of B. polymyxa FdI and FdII were estimated by gel filtration (8). Protein was determined by a modification of the phenol method (9) with bovine serum albumin as a standard. Nonheme (ferrous) iron was determined by the o-phenanthroline method of Harvey et al. (10) as modified by Lovenberg et al. (9). Inorganic sulfide was determined by the method of Fogo and Popowsky (11) as adapted to ferredoxin (9). Polyacrylamide gel electrophoresis was carried out at room temperature using 20% acrylamide (Tris-glycine buffer, pH 8.3) (12). The amino acid composition was determined by the standard methods of Moore and Stein (13). Biochemical assays. The effectiveness of the two B. polymyxa ferredoxins as electron carriers ws.s measured by their ability to mediate electron transfer between illuminated spinach chloroplast nitrogenase by methfragments and B. polymyxa ods previously described for A. vinelandii ferredoxins (4, 14). Ferredoxin-free chloroplast fragments were prepared as previously described (15) and nitrogenase activity was measured by reduction of acetylene to ethylene (16,17) as determined by gas chromatography. Biological activities of B. polymyxa FdI and FdII were also assayed by their ability to replace spinach ferredoxin in the photoreduction of NADP by ferredoxin-free chloroplasts as described by Buchanan and Arnon (18). RESULTS
Two ferredoxins
AND DISCUSSION
from B.
cellulose chromatography
polymyxa. DEAEof the crude ferre-
TWO FERREDOXINS
FROM Racillug polymy.rcc
Fraction
FIG. 1. Chromatography of B. polymyxa ferredoxins on DEAE-cellulose. The column (1.5 X 35 cm) was eluted with 0.02 M phosphate buffer, pH 7.4, containing NaCl as indicated. Collection of 4.5-ml fractions began just before elution of FdII from column.
cut from the gel, eluted, and shown to still doxin preparation obtained from B. polyhave biological activity. myxa resulted in the elution of two brown The spectra of purified FdI and FdII apferredoxin bands (Fig. 1). The ferredoxin elating at the high salt concentration (0.35 M pear to be quite similar; they have absorpNaCl) was FdI, while that eluting at a lower tion maxima in the visible region of the specsalt concentration (0.28 M SaCl) was FdII. trum at 385 nm and a peak in the ultraviolet FdI was the species previously purified and at 280 nm (Fig. 3). The spectrum of FdII had characterized (5-7). The relative abundance to be taken within several hours after t,he of FdI and FdII varied with each preparaprotein eluted from the column as the 350- to tion, probably because FdII slowly deterio420-nm region of the spectrum deteriorated rated under the aerobic purification condiwith time under aerobic conditions (dashed tions. (The stability of FdII is discussed line in Fig. 3). below.) FdII was observed to be as much as The absorbance ratio of 0.54-0..55 for l:dI 80% and as little as 20% of the total ferre- and freshly prepared FdII was taken as an doxin yield. The amount of FdI appeared to indication of purity because further purificaremain fairly constant from one preparation tion attempts resulted in an identical ab to another. sorbance ratio of prosthetic group (L&,) to While DEAE-cellulose chromatography protein (A280) and, furthermore, the same Jeparated the two ferredoxins, Sephadex value was calculated from the ferrcdoxin chromatography showed a single ferredoxin spectrum reported by Shethna et al. (5). Chemical composition ad molecular weight. band eluting from the column, suggesting ::ither that the t,wo ferredoxins had near A comparison of the amino acid conlposit& dcntical molecular weights or that they were of FdI and FdII clearly shows thc>m to be ‘orming a complex. Polyacrylamide gel elec- distinct chemical species (Table I). E’dI has a :rophoresis of this single brown band from total of 23 acidic amino acid residues, while Sephadex showed it’ to be a mixture of the FdII has 18 acidic residues, a difference ap,wo ferredoxins (Fig. 2), indicating similar parently large enough to account for the nolecular weights (see below). For identifiseparation of these> two ferredoxins on :ation of FdI and FdII on polyacrylamide DEAE-cellulose and polyacrylamidc gel :els, samples of the two ferredoxins previelectrophoresis. Asparagine and glutaminc ~sly separated on DEAE-cellulose were run were not dist’inguished from aspartic and s standards (Fig. 2). Bacillus polymyxa FdI glutamic acid. Comparing the amino acid nigrates as a sharp band, while the FdII composition of FdI with that of the four land is somewhat hazy. As these gels were iron-sulfur B. polymyxa ferredoxin \v(’ charlot stained, the ferredoxin bands could be act’erized earlier (6) shows them to be the
636
YOCH
t
Fd I Sephadex
Fd4
DEAE-cellulose
FIG. 2. Polyacrylamide gel electrophoretic patterns of l?. polymyxa ferredoxin from Sephadex G-50 and DEAE-cellulose. Gels were not stained as the ferredoxins be identified by their brown color.
same protein. The amino acid composition of another four iron-sulfur ferredoxin (from Desulfovibrio gigas) (19) is similar to that of B. polymyxa FdI. As FdI is the stable species of B. polymyxa, it is tempting to speculate on the existence of a second, perhaps unstable, species of ferredoxin in D. gigas analogous to B. polymyxa FdII. The partial specific volume of FdI was calculated from the amino acid composition (Table I) to be 0.671 cc/g (for the methods, see Ref. 3). The molecular weights of FdI and FdII estimated by gel filtration (Sephadex G-100) were both approximately 7800 (Fig. 4). As a protein marker with a molecular weight less than 7000 was not available, these values may be subject to some degree of error as the standard curve is not linear in this region. However, a molecular weight of 7800 is in general agreement with the value of “near 9000” reported for B. polymyxa ferredoxin (FdI) by Shethna et al. (5). Estimation of molecular weight of these ferredoxins on Sephadex G-50 columns, however, consistently gave values of 16,00018,000 (chromatography in the presence or
bands could
III 300
400 wavelength
500
FIG. 3. Absorption spectra of B. polymyxq ferredoxins I and II. The dashed line under FdII is the spectrum (in the visible region) of partially deteriorated FdII. All three proteins were in 0.05 M Tris-HCl buffer, pH 7.4, at a concentration of approximately 0.5 mg/ml.
TWO TABLE
FERREDO~XINS
PdII
FdI
Racillus
637
polymyxa
Although the amino acid composition of FdI and FdII differ, their iron-sulfur content appeared to be the same (Table II). Both of the ferredoxins appear to contain
I
AMINO ACID COMPOSITION OF Bacillus polymyxa FdI AND FdIp Amino acid
FROM
Resi- ’ Nearest integer duesh Per mole ! Lysine Histidine Arginine Tryptophan Aspartic acid Threonine Serine Gllltamic aci( 3 Proline Glycine Alanine Half-cyst.ine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
3.73 0.17 1.39 n.d.c 15.32 5.33 2.40 8.34 3.65 6.01 9 .34 2.91'6 3.12 0.50 5.54 3.52 3.51 2.27
14 0 1 1 1 15 5 ~ 3 ~ 8 1 4 6
Residuesh her mole
Yeares t integer
5.7G 0.37 0.51 n.d.< 9.22 3.10 5.74 8.78 2.95 7.76 7.16 2.41" 6.18 0.71 4.20 4.86 1.75 4.53
6 0 1
Tot al residue: 5
9 3 6 9 3 8 ,a, 6 1 4 5 2 5
79
‘&The results are the average of duplicate analyses of samples hydrolyzed 36 hr. h Based on a molecular weight of 8800. r Not determined. I’ Corrected for the loss of half-cystine during acid hydrolysis, assuming that the percent lost was the same as that lost during hydrolysis of Closlridiwrl acidi-urici ferredoxin.
absence of 1 M KaC1).2 These values were considered anomalous because they did not
agree with molecular weight estimations from sedimentation coefficients (1.3-1.4) obtained
from
ccntrifugation
analysis
of the
ferredoxins. The minimum molecular weights calculated from the amino acid compositions (SW Tahk I) \+w-c approximately 8800. Regardless of the method of determination, the molecular weights of FdI and FdII were always idrnt,iral. 2 Sephadex elut ion volumes of clostridial ferredoxins vary considerably, depending on the sodium chloride concentration of the eluting buffer (personal communication from Dr. Eglis T. Lode).
Fd
115 jg s IIOc .o I ; 105’ t100 c
;-7&iT5,000
10,000
,
Molecular
Weight
G&iii
weight estimation of U. FIG. 4. Molecular polymyxa ferredoxins I and II by gel filtration. Protein markers (14 mg of each protein in 2.0 ml of 0.02 M phosphate bsffer, pH 7.4) were applied to a Sephadex G-100 column (1.5 X 90 cm). The ferredoxins (approximately 4 mg) were applied separately in t,he same volume of buffer a.s 1he marker proteins. The column was ehlted with 0.02 M phosphate buffer, pH 7.4. Elution volume of the ferredoxins was determined by their absorbance at 385 nm. TABLE
II
SOMIC PILOPERTIES OF Bacillws polymgxa FERRKDOXINS FdI Nonheme irona Acid-labile sulfura Half-cystineb Molecular weight A 38onm/A28onrn
4 4 4 7800~ 88ood 0.55
FdII 3-i 3-4 -l 7700( 88OW 0.54-0.55
a Based on a molecular weight of 8800 with the protein concentration determined by the Folinphenol method. Variation in FdII may have been due to unknown amounts of denatured protein in the sample; see earlier discussion of FdII stability. b See Table I. c Determined by gel filtration (Sephadex ClOO). d Minimum molecular weight est.imated from a.mino acid composition.
638
YOCH
8. po&myxa
g 0.08
0 FdI FdII
G - 0.06 -a 0) s 2 0.04 a 2 a z
l
l
0.02 ,!;i
oLd0.015LLLLI 0.030 0.045 Fertedoxin Concentration (absorbance at 385 nm)
FIG. 5. Activity of B. polymyxa ferredoxins I and II in the photochemical reduction of NADP by chloroplasts. The complete reaction mixture contained, in 1.0 ml, ferredoxin-free spinach chloroplasts (50 rg of chlorophyll) and the following (in moles) : Tricine (N-Tris (hydroxymethyl)methyl-glycine) buffer, pH 8.0, 100; ascorbate, 10; 2,6-dichlorophenol indophenol, 0.05; MgCln, 5.0; NADP, 2.0; and B. polymyza FdI or FdII as indicated. Ferredoxin concentration was determined as a function of absorbance at 385 nm (d = 1 cm) in the reaction mixture. Light intensity, 9OWl fc.
four nonheme iron and four acid-labile sulfur atoms per mole, based on a molecular weight of 8800. FdII tended to give iron-sulfur values of less than four (atoms/mole) but this was probably due to the presence of some inactive protein at the time of analysis. Biological activities. Bacillus polymyxa ferredoxin (FdI) was previously reported to substitute for the two iron-sulfur plant ferredoxin in the photochemical reduction of NADP by spinach chloroplasts (6). Figure 5 shows that FdI and FdII were equally effective in this reaction when compared on the basis of absorbance at 385 nm (the absorbance maxima of the active iron-sulfur prosthetic group). The ability of these electron carriers (FdI and FdII) to link the Bacillus nitrogenase system to photochemically generated reducing power of spinach chloroplasts is shown in Fig. 6. There is no immediate explanation for FdII being more effective as a reductant
for nitrogenase under these conditions than is Fdl. While nitrogenases from Chromatium (20), A. vinelandii (unpublished results), and other bacteria (see review, 21) do not couple effectively with spinach ferredoxin, B. polymyxa nitrogenase was equally active with photoreduced spinach ferredoxin or its native FdII (Fig. 7). This observation suggests a similarity in the active site of the Bacillus four iron-sulfur ferredoxin and the two ironsulfur plant ferredoxin. Such a similarity has already been suggested from electron paramagnetic resonance (EPR) studies of reduced B. polymyxa ferredoxin (FdI), as its EPR spectrum closely resembles that of the reduced plant ferredoxin (5). Furthermore, it has been suggested that the B. polymyxa ferredoxin utilizes one of the two iron-sulfur centers “for obligate 1 e- transfer, a property
Ferredoxin (absorbance
Concentration at 385 nm)
FIG. 6. Effectiveness of the two B. pol2/myxa ferredoxins in the nitrogenase reaction. The complete system contained, in 1.5 ml, ferredoxinfree spinach chloroplasts (300 pg chlorophyll), B. polymyxa nitrogenase, 8.0 mg, and the following (in &moles) : HEPES (iV-2-hydroxyethylpiperasine-l\r’-2-ethane sulfonic acid) buffer, pH 7.4, 50; ascorbate, 10; 2,6-dichlorophenol indophenol, 0.05; MgClZ, 5; creatine phosphate, 40; ATP, 4, and creatine phosphokinase, 0.01 mg; and B. polymyxa FdI or FdII as indicated. Ferredoxin concentration was determined as a function of absorbance at 385 nm (d = 1 cm) in the reaction mixture. Light intensity, 9006 fc; gas phase, 73oj, argon + 27% acetylene; temperature, 30°C.
results). Comparing the activities of the t’no B. polymyza. ferredoxins in these various ferredoxin-dependent reactions may help to clarify the situation.
1
‘i
J-3
6 UM
FIG. 7. Effectiveness of photochemically reduced U. polgm:tyxa FdII and spinach ferredoxin in the U. polllmqsn nitrogenase reaction. Reaction mixture and conditions are described in the legend of Fig. 6 with ferredoxin concentrations as indicated.
in common \vit’h plant-type iron-sulfur prot,eins” (7). On the other hand, the spectra of the B. polgmyxa fcrredoxins, lvith their peak in the visible rt,gion at 385 nm, and their brown coior, suggest similarities to the eight ironsulfur hactcrial fcrredoxins. Thus t’he four iron-sulfur Bacillus fcrredoxins indeed appcaar to bc int,crmediate bct’lvcen the plant and bac*t,orial ferredoxins. Why nn organism like B. polymyza, lvhich is alosc1y related to the elostridia (22, 23) should have t,n-o fcrredoxins is not understood at this time. Thr greater effectiveness of l:dlI compared to FdI in the nitrogenase reaction may suggest that one fcrredoxin is +pc&ic for one clnzymc, lvhile the other ferredoxin is specific for a sc>condenzyme (hydrocenasc, for cxamplc). If such specificity does .lxist, it may bc due to the physical separation of several ft,rrcdoxin-dependent ens.yrncxs. Icor c>xamplc, in B. polymyza the jyruvatct dchgdrogcnase (a hydrogcn-donatng system for nit.rogcnase) (24) and the YADPH-fcrredoxin reductasc (25) are solu)lc, \vhil(l the hydrogcnase (24) and a fornatn hydrogen-donating system (also linked o nitrogc:nascx) RIY particulate> (unpublished
,Vote added i-n proof. While the manuscript was in preparation, the author became aware that, N. A. Stombaugh and W. H. Orme-Johnson (Ijepartment of Biochemist,ry, University of Wisconsin) had also sltbmitted a manuscript describing the two ferredoxins in Kndlus polyn~yra; the designations for the t,wo ferredoxins as FdI and FdII are consistent. in the two papers. ACKh’OWLEDGMENT: 1 am indebted to I)r. ICglis T. Lode chemistry Department, University of Berkeley, for helpful discrlssions during of this investigation and for doing t.he analysis and to Dr. D. I. Brnon for help in the preparation of the manllscript.
of the BioCalifornia, the course amino acid and advice
1. SH\NMUGAM, E<. T., .\ND .kRNON, 1). 1. (l%l) Fed. Proc. 30, 1136. 2. SHANMUG:~M, K. T., BucH.\N.\N, B. B., .\x~D AltsoX, D. I. (1972) Ir’iochi~~ Biophys. Aclo 256, 477-186. I. (l'J72) ,/. Iii/d. 3. YOCH, 1). c., ASU kUXO?;;i). Chem. 24’7, 451-1-1520.
4.
YOCH,
1). c.,
BIGJGMANN,
J.
It.,
\‘ALJWTNI,,
Ii. C., .\pin AHNON, 1). 1. (1969) l’roc. .\.nt. Actcd. Sci. USA 64, l-10&1410. 5. SHKTHXA, Y. I., STOMLUUGH, ?;. A., ASD Bumt~s, It. II. (1971) h’iochenl, Liio~h+~ Res. Con,,tnu,l. 42, 1108-1116. 6. YOCH, 1). c., .\NI) ~lLbXTINi7, 1:. ('. (1972) J. Uuc/eriol. 110, 1211-1213. 7. OKM~-JOHSSON, W. H., STOMII.\LYGH, N. A, ANI) BL~RIS, It. H. (1972) Fed. P,oc. 31, 448. 8. ANDKE~S, P. (1965) hiochenr. J. 96, 5!kWiOC,. 9. LOVIiNHl-RG, KUUNOWITZ,
w.,
b2HAXA5,
B.
I!,.,
.\ND
J. (‘. (1963) J. Rio!. C’henl. 238,
3899-3913. 10. H.\avIX, A. I’., JR., SMART, J. A., .\xn A~lb, 1;. S. (1955) AM&Z. Chen‘. 27, Z-35. 11. Foco, J. K., ASD PoPo\\-SI
14. BENEM.~SK., J. I:., YocH, 1). C., \'.\I.I~,.uTIwI~;, IL. c’., ANI) A~XON, D. I. (1969) I’roc. .\‘clt. Acud. Sci. USA 64, 1079-1086. 15. YOCH, 1). c:. (1972) Bi0CheWz. %0&/s. &!S. Conl?rm,/. 49, 3s5-:S2.
640
YOCH
16. SCH~~LLHORN, R., AND BURRIS, R. H. (1966) Fed. Proc. 26, 710. 17. DILWORTH, M. J. (1966) Biochim. Biophys. Acta 137, 265-294. 18. BUCHANAN, B. B., AND ARNON, D. I. (1971) Methods Enzymol. 23,4X%440. 19. LAISHLEY, E. J., TRAVIS, J., AND PECK, H. D., JR. (1969) J. Bacterial. 98, 302-303. 20. YOCH, D. C., AND ARNON, D. I. (1970) Biochim. Biophys. Acta 197, 180-184.
21. HARDY, R. W. F., AND BURNS, R. C. in IronSulfw Proteins (Lovenberg, W., ed.), Academic Press, N. Y., in press. 22. HINO, S., AND WILSON, P. W. (1958) J. Bacteriol. 76, 403408. 23. WITZ, D. F., DETROY, R. W., P. W. (1967) Arch. Mikrobiol. 24. GRAU, F. H., AND WILSON, P. Bacterial. 86, 446450. 25. YOCH, D. C. (1973) J. Bacterial.
AND WILSON, 66, 369-381.
W. (1963) J. in press.
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
and
633-640 (1973)
168,
Properties
Nitrogen-Fixing
of Two
Bacterium DUANE
Department
of Cell Physiology,
Ferredoxins
Bacillus
from
the
polymyxa'
C. YOCH
University
of California,
Received
Berkeley,
California
94720
May 3, 1973
Two ferredoxins, designated FdI and FdII, have been isolated from the nitrogenfixing bacterium Bacillus polymyza. The two ferredoxins were readily separated on DEAE-cellulose and disc gel electrophoresis. The amino acid compositions of FdI and FdII showed them to be different protein species; the greater number of acidic amino acid residues in FdI than in FdII appears to account for separation based on electronic charge. FdI and FdII were both found to have four nonheme iron and folu acid-labile sulfur groups per mole. The absorption spectra of the two ferredoxins are almost identical, with a peak in the visible region of the spectrum at 385 nm. The Aass/A~so absorbance ratio of both ferredoxins was 0.540.55. FdII was not stable under aerobic conditions, as indicated by a decrease in the visible region of the spectrum. Both FdI and FdII have nearly identical molecular weights, as judged by gel filtration and amino acid composition (approx 8800). The purified ferredoxins catalyzed the photoreduction of NADP by spinach chloroplasts with equal effectiveness. In the nitrogen-fixation reaction of B. polymyxcz, FdII was more effect.ive than FdI.
Recent studies in this laboratory on bacterial ferredoxins have shown that several species of bacteria have not one but two biologically active ferredoxins. Rhodospirillum rubrum, a facultative phototroph, was the first organism from which two types of ferredoxin were isolated (1, 2). These ferredoxins, referred to as types I and II (FdI and FdII), differed in iron-sulfur content, amino acid composition, spectra, and molecular weight. This was the first demonstration that ferredoxins, like cytochromes, can occur in the cell in several forms. A second organism from which two biologically active ferredoxins were isolated is the nitrogen-fixing obligate aerobe Axotobatter vinelandii (3). The two ferredoxins (designated azotobacter FdI and FdII) differed in spectral properties, oxidationreduction potential, and size. Both ferrer Part of this work was presented in a paper given at the Dinitrogen (NJ Fixation Seminar at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, May 9, 1973. 633 2opyright Ul rights
@ 1973 by Academic Preen, of reproduction in any form
Inc. reserved.
doxins from Azotobacter functioned in the nitrogenase reaction of that organism and were able to replace spinach ferredoxin in the photochemical reduction of NADP by spinach chloroplast fragments (3, 4). In the course of an investigation aimed at characterizing a ferredoxin (5) from the nitrogen-fixing bacterium Bacillus polymyxa, Yoch and Valentine (6) made the preliminary observation that this organism, like R. rubrum and A. vinelandii, contained tlvo ferredoxins. These ferredoxins were distinguished from one another by their elution characteristics on DEAE-cellulose; tn-o biologically active ferredoxin fractions were obtained. Further purification of the recombined ferredoxin fractions resulted in the isolation of only one species of (near homogeneous) ferredoxin. It had the unusual property of containing four atoms of nonhcme iron and acid-labile sulfur per molecule (6, 7) making it an intermediate form compared to the eight iron-sulfur bacterial ferredoxins and the two iron-sulfur plant ferredoxins. The fate of the second ferredoxin was a mat,-
634
YOCH
ter of speculation
in the preliminary report (6). This paper presents evidence that two chemically distinct species of ferredoxin (designated FdI and FdII) exist in B. po&myza. FdI corresponds to the protein initially described by Shethna et al. (5) and later characterized by Orme-Johnson et al. (7) and Yoch and Valentine (6) ; FdII is the protein initially reported to be lost during the purification process (6). These ferredoxins differ in amino acid composition, biological activity in the B. polymyxa nitrogenase reaction, and stability in air (FdII is oxygen-labile). They appear to be identical in iron-sulfur content, molecular weight, and spectral characteristics. METHODS Culture. Bacillus polymyxa strain Hino (obtained from Dr. R. V. Klucas of the University of Nebraska) was grown in 12-liter carboys in medium containing (g/liter) : K~HP04.3Ht0, 14.3; KH2POI, 11.8; CaC12.2Ht0, 0.05; MgSOd, 0.25; N&l, 0.01; FeS04.7Hz0, 0.015; Na2Mo04. 2H20, 0.005; biotin, 0.00005; sucrose, 20; and (NH4)*S04, 2.0, as nitrogen source. NO precautions were taken to make the cultures anaerobic other than filling the carboys to the neck and covering them with aluminum foil. After 24-36 hr (29-31”C), the cultures were harvested and used for the isolation of ferredoxin. Nitrogen-fixing cultures were obtained by omitting the (NH4)$04 from the medium and sparging the culture with Nz gas. Isolation of jerredoxin. Cells (1500 g wet wt) grown on the (NH4)2S04 medium were suspended in 3 vol of 0.02 M phosphate buffer, pH 7.4, and disrupted by sonication for 5 min at 5°C with a 20.kHz Branson sonifier at full power. The disrupted cells and debris were incubated with 2 mg of deoxyribonuclease at room temperature for 1 hr and then centrifuged at 32,OOOgfor 10 min. The resulting supernatant. fluid was passed over a bed of DEAE-cellulose (6 X 20 cm) equilibrated with 0.02 M phosphate buffer, pH 7.4. This buffer was used throughout the purificat.ion process. The ferredoxin was adsorbed at the top of the column as a brown band. The column was washed with loo-ml portions of buffer containing no salt, 0.1 M NaCl, and 0.2 M NaCl. The ferredoxin was eluted from the column with buffer containing 0.4 M NaCl; the ferredoxin solution was then dilut,ed fivefold and charged on a DEAE-cellulose chromatography column (1.5 X 35 cm). Discontinuous elution of the column with 75.ml portions of 0.28, 0.30, and 0.35 M NaCl in phosphate buffer
showed two distinct ferredoxin bands eluting from the column. From this point on, the two ferredoxins were each purified separately, although identical procedures were used. FdII was stored under argon between purification steps. The ferredoxins (buffered with 0.1 M phosphate buffer, pH 7.4) were further purified by ammonium sulfate fractionat,ion. The colorless precipitates that formed between 0 and 65% ammonium sulfate saturation were removed by centrifugation (35,000g for 10 min) and discarded. The ferredoxins were then precipitated by the further addition of ammonium sulfate to 100% saturation, the pellets were dissolved in 2.0 ml of buffer and were individually chromatographed on a Sephadex G-100 column (1.5 X 85 cm). Fractions with absorbance ratios A&A280 between 0.54 and 0.55 were pooled and used for characterization of the two ferredoxins . Although the ferredoxins isolated, purified, and characterized in this study were from NH,+grown cells, the same two ferredoxins were also isolated from Nz-grown cultures of B. polymyza. Analytical procedures. The molecular weights of B. polymyxa FdI and FdII were estimated by gel filtration (8). Protein was determined by a modification of the phenol method (9) with bovine serum albumin as a standard. Nonheme (ferrous) iron was determined by the o-phenanthroline method of Harvey et al. (10) as modified by Lovenberg et al. (9). Inorganic sulfide was determined by the method of Fogo and Popowsky (11) as adapted to ferredoxin (9). Polyacrylamide gel electrophoresis was carried out at room temperature using 20% acrylamide (Tris-glycine buffer, pH 8.3) (12). The amino acid composition was determined by the standard methods of Moore and Stein (13). Biochemical assays. The effectiveness of the two B. polymyxa ferredoxins as electron carriers ws.s measured by their ability to mediate electron transfer between illuminated spinach chloroplast nitrogenase by methfragments and B. polymyxa ods previously described for A. vinelandii ferredoxins (4, 14). Ferredoxin-free chloroplast fragments were prepared as previously described (15) and nitrogenase activity was measured by reduction of acetylene to ethylene (16,17) as determined by gas chromatography. Biological activities of B. polymyxa FdI and FdII were also assayed by their ability to replace spinach ferredoxin in the photoreduction of NADP by ferredoxin-free chloroplasts as described by Buchanan and Arnon (18). RESULTS
Two ferredoxins
AND DISCUSSION
from B.
cellulose chromatography
polymyxa. DEAEof the crude ferre-
TWO FERREDOXINS
FROM Racillug polymy.rcc
Fraction
FIG. 1. Chromatography of B. polymyxa ferredoxins on DEAE-cellulose. The column (1.5 X 35 cm) was eluted with 0.02 M phosphate buffer, pH 7.4, containing NaCl as indicated. Collection of 4.5-ml fractions began just before elution of FdII from column.
cut from the gel, eluted, and shown to still doxin preparation obtained from B. polyhave biological activity. myxa resulted in the elution of two brown The spectra of purified FdI and FdII apferredoxin bands (Fig. 1). The ferredoxin elating at the high salt concentration (0.35 M pear to be quite similar; they have absorpNaCl) was FdI, while that eluting at a lower tion maxima in the visible region of the specsalt concentration (0.28 M SaCl) was FdII. trum at 385 nm and a peak in the ultraviolet FdI was the species previously purified and at 280 nm (Fig. 3). The spectrum of FdII had characterized (5-7). The relative abundance to be taken within several hours after t,he of FdI and FdII varied with each preparaprotein eluted from the column as the 350- to tion, probably because FdII slowly deterio420-nm region of the spectrum deteriorated rated under the aerobic purification condiwith time under aerobic conditions (dashed tions. (The stability of FdII is discussed line in Fig. 3). below.) FdII was observed to be as much as The absorbance ratio of 0.54-0..55 for l:dI 80% and as little as 20% of the total ferre- and freshly prepared FdII was taken as an doxin yield. The amount of FdI appeared to indication of purity because further purificaremain fairly constant from one preparation tion attempts resulted in an identical ab to another. sorbance ratio of prosthetic group (L&,) to While DEAE-cellulose chromatography protein (A280) and, furthermore, the same Jeparated the two ferredoxins, Sephadex value was calculated from the ferrcdoxin chromatography showed a single ferredoxin spectrum reported by Shethna et al. (5). Chemical composition ad molecular weight. band eluting from the column, suggesting ::ither that the t,wo ferredoxins had near A comparison of the amino acid conlposit& dcntical molecular weights or that they were of FdI and FdII clearly shows thc>m to be ‘orming a complex. Polyacrylamide gel elec- distinct chemical species (Table I). E’dI has a :rophoresis of this single brown band from total of 23 acidic amino acid residues, while Sephadex showed it’ to be a mixture of the FdII has 18 acidic residues, a difference ap,wo ferredoxins (Fig. 2), indicating similar parently large enough to account for the nolecular weights (see below). For identifiseparation of these> two ferredoxins on :ation of FdI and FdII on polyacrylamide DEAE-cellulose and polyacrylamidc gel :els, samples of the two ferredoxins previelectrophoresis. Asparagine and glutaminc ~sly separated on DEAE-cellulose were run were not dist’inguished from aspartic and s standards (Fig. 2). Bacillus polymyxa FdI glutamic acid. Comparing the amino acid nigrates as a sharp band, while the FdII composition of FdI with that of the four land is somewhat hazy. As these gels were iron-sulfur B. polymyxa ferredoxin \v(’ charlot stained, the ferredoxin bands could be act’erized earlier (6) shows them to be the
636
YOCH
t
Fd I Sephadex
Fd4
DEAE-cellulose
FIG. 2. Polyacrylamide gel electrophoretic patterns of l?. polymyxa ferredoxin from Sephadex G-50 and DEAE-cellulose. Gels were not stained as the ferredoxins be identified by their brown color.
same protein. The amino acid composition of another four iron-sulfur ferredoxin (from Desulfovibrio gigas) (19) is similar to that of B. polymyxa FdI. As FdI is the stable species of B. polymyxa, it is tempting to speculate on the existence of a second, perhaps unstable, species of ferredoxin in D. gigas analogous to B. polymyxa FdII. The partial specific volume of FdI was calculated from the amino acid composition (Table I) to be 0.671 cc/g (for the methods, see Ref. 3). The molecular weights of FdI and FdII estimated by gel filtration (Sephadex G-100) were both approximately 7800 (Fig. 4). As a protein marker with a molecular weight less than 7000 was not available, these values may be subject to some degree of error as the standard curve is not linear in this region. However, a molecular weight of 7800 is in general agreement with the value of “near 9000” reported for B. polymyxa ferredoxin (FdI) by Shethna et al. (5). Estimation of molecular weight of these ferredoxins on Sephadex G-50 columns, however, consistently gave values of 16,00018,000 (chromatography in the presence or
bands could
III 300
400 wavelength
500
FIG. 3. Absorption spectra of B. polymyxq ferredoxins I and II. The dashed line under FdII is the spectrum (in the visible region) of partially deteriorated FdII. All three proteins were in 0.05 M Tris-HCl buffer, pH 7.4, at a concentration of approximately 0.5 mg/ml.
TWO TABLE
FERREDO~XINS
PdII
FdI
Racillus
637
polymyxa
Although the amino acid composition of FdI and FdII differ, their iron-sulfur content appeared to be the same (Table II). Both of the ferredoxins appear to contain
I
AMINO ACID COMPOSITION OF Bacillus polymyxa FdI AND FdIp Amino acid
FROM
Resi- ’ Nearest integer duesh Per mole ! Lysine Histidine Arginine Tryptophan Aspartic acid Threonine Serine Gllltamic aci( 3 Proline Glycine Alanine Half-cyst.ine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
3.73 0.17 1.39 n.d.c 15.32 5.33 2.40 8.34 3.65 6.01 9 .34 2.91'6 3.12 0.50 5.54 3.52 3.51 2.27
14 0 1 1 1 15 5 ~ 3 ~ 8 1 4 6
Residuesh her mole
Yeares t integer
5.7G 0.37 0.51 n.d.< 9.22 3.10 5.74 8.78 2.95 7.76 7.16 2.41" 6.18 0.71 4.20 4.86 1.75 4.53
6 0 1
Tot al residue: 5
9 3 6 9 3 8 ,a, 6 1 4 5 2 5
79
‘&The results are the average of duplicate analyses of samples hydrolyzed 36 hr. h Based on a molecular weight of 8800. r Not determined. I’ Corrected for the loss of half-cystine during acid hydrolysis, assuming that the percent lost was the same as that lost during hydrolysis of Closlridiwrl acidi-urici ferredoxin.
absence of 1 M KaC1).2 These values were considered anomalous because they did not
agree with molecular weight estimations from sedimentation coefficients (1.3-1.4) obtained
from
ccntrifugation
analysis
of the
ferredoxins. The minimum molecular weights calculated from the amino acid compositions (SW Tahk I) \+w-c approximately 8800. Regardless of the method of determination, the molecular weights of FdI and FdII were always idrnt,iral. 2 Sephadex elut ion volumes of clostridial ferredoxins vary considerably, depending on the sodium chloride concentration of the eluting buffer (personal communication from Dr. Eglis T. Lode).
Fd
115 jg s IIOc .o I ; 105’ t100 c
;-7&iT5,000
10,000
,
Molecular
Weight
G&iii
weight estimation of U. FIG. 4. Molecular polymyxa ferredoxins I and II by gel filtration. Protein markers (14 mg of each protein in 2.0 ml of 0.02 M phosphate bsffer, pH 7.4) were applied to a Sephadex G-100 column (1.5 X 90 cm). The ferredoxins (approximately 4 mg) were applied separately in t,he same volume of buffer a.s 1he marker proteins. The column was ehlted with 0.02 M phosphate buffer, pH 7.4. Elution volume of the ferredoxins was determined by their absorbance at 385 nm. TABLE
II
SOMIC PILOPERTIES OF Bacillws polymgxa FERRKDOXINS FdI Nonheme irona Acid-labile sulfura Half-cystineb Molecular weight A 38onm/A28onrn
4 4 4 7800~ 88ood 0.55
FdII 3-i 3-4 -l 7700( 88OW 0.54-0.55
a Based on a molecular weight of 8800 with the protein concentration determined by the Folinphenol method. Variation in FdII may have been due to unknown amounts of denatured protein in the sample; see earlier discussion of FdII stability. b See Table I. c Determined by gel filtration (Sephadex ClOO). d Minimum molecular weight est.imated from a.mino acid composition.
638
YOCH
8. po&myxa
g 0.08
0 FdI FdII
G - 0.06 -a 0) s 2 0.04 a 2 a z
l
l
0.02 ,!;i
oLd0.015LLLLI 0.030 0.045 Fertedoxin Concentration (absorbance at 385 nm)
FIG. 5. Activity of B. polymyxa ferredoxins I and II in the photochemical reduction of NADP by chloroplasts. The complete reaction mixture contained, in 1.0 ml, ferredoxin-free spinach chloroplasts (50 rg of chlorophyll) and the following (in moles) : Tricine (N-Tris (hydroxymethyl)methyl-glycine) buffer, pH 8.0, 100; ascorbate, 10; 2,6-dichlorophenol indophenol, 0.05; MgCln, 5.0; NADP, 2.0; and B. polymyza FdI or FdII as indicated. Ferredoxin concentration was determined as a function of absorbance at 385 nm (d = 1 cm) in the reaction mixture. Light intensity, 9OWl fc.
four nonheme iron and four acid-labile sulfur atoms per mole, based on a molecular weight of 8800. FdII tended to give iron-sulfur values of less than four (atoms/mole) but this was probably due to the presence of some inactive protein at the time of analysis. Biological activities. Bacillus polymyxa ferredoxin (FdI) was previously reported to substitute for the two iron-sulfur plant ferredoxin in the photochemical reduction of NADP by spinach chloroplasts (6). Figure 5 shows that FdI and FdII were equally effective in this reaction when compared on the basis of absorbance at 385 nm (the absorbance maxima of the active iron-sulfur prosthetic group). The ability of these electron carriers (FdI and FdII) to link the Bacillus nitrogenase system to photochemically generated reducing power of spinach chloroplasts is shown in Fig. 6. There is no immediate explanation for FdII being more effective as a reductant
for nitrogenase under these conditions than is Fdl. While nitrogenases from Chromatium (20), A. vinelandii (unpublished results), and other bacteria (see review, 21) do not couple effectively with spinach ferredoxin, B. polymyxa nitrogenase was equally active with photoreduced spinach ferredoxin or its native FdII (Fig. 7). This observation suggests a similarity in the active site of the Bacillus four iron-sulfur ferredoxin and the two ironsulfur plant ferredoxin. Such a similarity has already been suggested from electron paramagnetic resonance (EPR) studies of reduced B. polymyxa ferredoxin (FdI), as its EPR spectrum closely resembles that of the reduced plant ferredoxin (5). Furthermore, it has been suggested that the B. polymyxa ferredoxin utilizes one of the two iron-sulfur centers “for obligate 1 e- transfer, a property
Ferredoxin (absorbance
Concentration at 385 nm)
FIG. 6. Effectiveness of the two B. pol2/myxa ferredoxins in the nitrogenase reaction. The complete system contained, in 1.5 ml, ferredoxinfree spinach chloroplasts (300 pg chlorophyll), B. polymyxa nitrogenase, 8.0 mg, and the following (in &moles) : HEPES (iV-2-hydroxyethylpiperasine-l\r’-2-ethane sulfonic acid) buffer, pH 7.4, 50; ascorbate, 10; 2,6-dichlorophenol indophenol, 0.05; MgClZ, 5; creatine phosphate, 40; ATP, 4, and creatine phosphokinase, 0.01 mg; and B. polymyxa FdI or FdII as indicated. Ferredoxin concentration was determined as a function of absorbance at 385 nm (d = 1 cm) in the reaction mixture. Light intensity, 9006 fc; gas phase, 73oj, argon + 27% acetylene; temperature, 30°C.
results). Comparing the activities of the t’no B. polymyza. ferredoxins in these various ferredoxin-dependent reactions may help to clarify the situation.
1
‘i
J-3
6 UM
FIG. 7. Effectiveness of photochemically reduced U. polgm:tyxa FdII and spinach ferredoxin in the U. polllmqsn nitrogenase reaction. Reaction mixture and conditions are described in the legend of Fig. 6 with ferredoxin concentrations as indicated.
in common \vit’h plant-type iron-sulfur prot,eins” (7). On the other hand, the spectra of the B. polgmyxa fcrredoxins, lvith their peak in the visible rt,gion at 385 nm, and their brown coior, suggest similarities to the eight ironsulfur hactcrial fcrredoxins. Thus t’he four iron-sulfur Bacillus fcrredoxins indeed appcaar to bc int,crmediate bct’lvcen the plant and bac*t,orial ferredoxins. Why nn organism like B. polymyza, lvhich is alosc1y related to the elostridia (22, 23) should have t,n-o fcrredoxins is not understood at this time. Thr greater effectiveness of l:dlI compared to FdI in the nitrogenase reaction may suggest that one fcrredoxin is +pc&ic for one clnzymc, lvhile the other ferredoxin is specific for a sc>condenzyme (hydrocenasc, for cxamplc). If such specificity does .lxist, it may bc due to the physical separation of several ft,rrcdoxin-dependent ens.yrncxs. Icor c>xamplc, in B. polymyza the jyruvatct dchgdrogcnase (a hydrogcn-donatng system for nit.rogcnase) (24) and the YADPH-fcrredoxin reductasc (25) are solu)lc, \vhil(l the hydrogcnase (24) and a fornatn hydrogen-donating system (also linked o nitrogc:nascx) RIY particulate> (unpublished
,Vote added i-n proof. While the manuscript was in preparation, the author became aware that, N. A. Stombaugh and W. H. Orme-Johnson (Ijepartment of Biochemist,ry, University of Wisconsin) had also sltbmitted a manuscript describing the two ferredoxins in Kndlus polyn~yra; the designations for the t,wo ferredoxins as FdI and FdII are consistent. in the two papers. ACKh’OWLEDGMENT: 1 am indebted to I)r. ICglis T. Lode chemistry Department, University of Berkeley, for helpful discrlssions during of this investigation and for doing t.he analysis and to Dr. D. I. Brnon for help in the preparation of the manllscript.
of the BioCalifornia, the course amino acid and advice
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14. BENEM.~SK., J. I:., YocH, 1). C., \'.\I.I~,.uTIwI~;, IL. c’., ANI) A~XON, D. I. (1969) I’roc. .\‘clt. Acud. Sci. USA 64, 1079-1086. 15. YOCH, 1). c:. (1972) Bi0CheWz. %0&/s. &!S. Conl?rm,/. 49, 3s5-:S2.
640
YOCH
16. SCH~~LLHORN, R., AND BURRIS, R. H. (1966) Fed. Proc. 26, 710. 17. DILWORTH, M. J. (1966) Biochim. Biophys. Acta 137, 265-294. 18. BUCHANAN, B. B., AND ARNON, D. I. (1971) Methods Enzymol. 23,4X%440. 19. LAISHLEY, E. J., TRAVIS, J., AND PECK, H. D., JR. (1969) J. Bacterial. 98, 302-303. 20. YOCH, D. C., AND ARNON, D. I. (1970) Biochim. Biophys. Acta 197, 180-184.
21. HARDY, R. W. F., AND BURNS, R. C. in IronSulfw Proteins (Lovenberg, W., ed.), Academic Press, N. Y., in press. 22. HINO, S., AND WILSON, P. W. (1958) J. Bacteriol. 76, 403408. 23. WITZ, D. F., DETROY, R. W., P. W. (1967) Arch. Mikrobiol. 24. GRAU, F. H., AND WILSON, P. Bacterial. 86, 446450. 25. YOCH, D. C. (1973) J. Bacterial.
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