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A molybdenum-containing (2Fe, 2S) protein from desulphovibrio gigas, a sulphate reducer
Journal of the Less-Common Metals, 54 (1977) 555 - 562 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
555
A MOLYBDENUM-CONT~~NG @Fe, 2s) PROTEIN FROM DESULPHOVIBRIO GIGAS, A SULPHATE REDUCER
J. J. G. MOURA and A. V. XAVIER Centro de Quimica Estruturat da Universidade de Lisboa, I.S.T., Lisbon-l {Portugal) M. BRUSC~I Labomtoire
and J. LEGALL de Chimie BactQritGzne, CNRS, 13274, Marseille, Cedex 2 (France)
J. M. P. CABRAL Labomtko
de Fisica e Engenharia Nucleares, Sacadm
(Portugal)
An acidic protein that contains molybdenum, iron and labile sulphide has been isolated from Desulphovibrio gigas, a sulphate reducer. The protein was purified by chromatographic procedures over DEAE-cellulose, Sephadex and alumina. The molecular weight, determined by polyacrylamide gel electrophoresis, is about 120 000. The amino acid composition is typical of the (ZFe, 2s) ferredoxins with a high propo~ion of acidic residues and about two cysteine residues per iron atom. It has twenty atoms of iron and twenty atoms of labile sulphide. One atom of molybdenum per molecule was determined by thermal neutron activation analysis. Optical absorption spectra indicate that the iron-sulphur chromophores are of the (2Fe, 2s) type.
Introduction Molybdenum has been shown to be present in different types of ironsulphur proteins. Examples of these complex iron-sulphur proteins are the molybdenum iron-sulphur flak hydroxylases (xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase and sulphite oxidase) which contain (2Fe, 2s) centres [ 11 and molybdenum-ferr~oxins where molybdenum is associated probably with (4Fe,4S) centres as sub-units of nitrogenase [2]. During the isolation of (4Fe,4S) ferredoxin from D. gigus we separated
*Presented at the Conference on “The Chemistry and Uses of Molybdenum”, University of Oxford, England, 31 August - 3 September, 1976, sponsored by Climax Molybdenum Co. Ltd. and the Chemical Society (Dalton Division).
556
R&we
mobilitv
Fig. 1. Electrophoretic polyacrylamide gel pattern (7.5% gel concentration) obtained on a single electrophoretic run. Seven proteins of known molecular weight were used: 1, cytochrome c, 12 460;2, ehymotrypsinogen A, 25 700; 3, egg_albumin, 46 000; 4 catalase, 60 000; 5, transferrin, 76 000; 6, Immunoglobulin G, 151000; 7, Immunoglobulm A, dim 360 000. References were run alone to confirm the assignments. Fig. 2. Plot of log (mol. wt.) (from data presented in Fig. 1) us. the electrophoretic mobility relative to bromophenol blue (polyacrylamide gel, polyacrylamide (7.5%), urea (3%), sodium dodecylsulphate (0.1%)).
another protein which proved to contain molybdenum and iron sulphur centres of the (2Fe,2S) type.
Experimental and results
Isolation of the (MO, Fe, S) protein
The isolation of this protein is based on its acidic characteristics. A frozen paste of cells (4 kg) was grown and treated as described previously [3]. All operations were carried at 0 - 4 “C unless otherwise stated. Tris-HCl and phosphate buffers (pH 7.6) of appropriate molarity were used. The fraction containing the (MO, Fe, S) protein was separated during the early stages of the purification of the three forms of the (4Fe, 45) ferredoxin [4]. The acidic extract [3] was adsorbed on a large DEAE-cellulose column and eluted with a discontinuous gradient 0.1 - 0.5 M Tris buffer. The fraction eluted with 0.2 - 0.3 M Tris buffer contains the (MO, Fe, S) protein together with flavoproteins, desulphoviridin and some cytochrome c3 (M.W., molecular
557
weight, 26 500) while the (4Fe, 4s) ferredoxin is eluted later (0.4 - 0.5 M Tris buffer). The (MO, Fe, S) protein was purified by rechromatography on DEAE-cellulose followed by columns of Sephadex G-50, silica gel and alumina. The purity of the protein was checked by polyacrylamide gel (Fig. 1) and has an absorbance ratio A2,s/A322 of 4.5. Molecular
weight
The molecular weight was estimated by polyacrylamide gel electrophoresis containing polyacrylamide (7.5%) in the presence of urea (3%) and sodium dodecylsulphate (0.1%) [ 51. The mobility of the (MO, Fe, S) protein was compared with seven proteins of known molecular weight used as standards and which gave the pattern shown in Fig. 1. A linear relationship between log (molecular weight) and the eiectrophoretic mobility referred to the dye (bromophenol blue) is obtained (see Fig. 2). A value of about 120 000 is estimated by this technique. The same value was obtained for the apoprotein which was produced after treatment with 3% HCl. No evidence of the presence of sub-units was found. Analysis of the constituents Molybdenum
of the protein
One atom of molybdenum per 120 000 mol. wt. was determined thermal neutron activation analysis.
by
Sample and standards. The sample was an aqueous solution of the (MO, Fe, S) protein (0.084 mM). Three aqueous standard solutions were prepared by dissolving NazMoO.+*2HzO p.a. in deionized water (2 X 10m5 M, 5 X 10M5 M and lo-*M). Aliquots (0.5 ml) of sample and standard solutions were introduced into about 5 ml quartz ampoules, cleaned beforehand in boiling HNO, and sealed at liquid nitrogen temperature under vacuum. Irradiation. The ampoules containing the sample and standards and an empty ampoule were irradiated for 14 h in the core grid of the RPI reactor (Sacavem) where the medium thermal neutron flux was 6.4 X 1012 n cmm2 s-l and the cadmium ratio (with cobalt) was 19.6. The set of ampoules was introduced into an aluminium can so that the ampoule containing the sample was symmetrically surrounded by the other four. Differences in neutron fluxes at the five positions at which the sample and standards stood were measured by means of 0.54% Co-Al alloy flux monitors. After allowing 24Na decay for 87 h, the irradiated substances were counted under exactly the same counting geometry. The counting time was between 2 and 5 h, depending on the concentration of molybdenum. Counting. The gamma-ray spectrometer consisted of a Ge( Li) coaxial detector (54 ml) connected through an Ortec model 120-4F preamplifier and an Ortec model 452 amplifier to an Inter-technique Didac 4000 multichannel analyser. This system had a resolution of about 2.5 keV for 1.333
558 TABLE 1 Characteristics of the D. gigas (MO, Fe, S) protein Molecular weight MO atoms/molecule Non-haem iron/molecule Labile sulphur/molecuIe Isoelectric point
120 000 1 - 20 - 20 4.1
MeV gamma rays. Calibration of the spectrometer was performed using standard gamma emitters. The spedrometric data were collected on punched tapes which were then processed in a PDP 15 computer using a computer program [6] which is an adap~tion of AKTAN [7]. The gamma spectrum of the irradiated ampoule containing the protein solution is shown in Fig. 3. For molybdenum determination, the 140.5 keV photopeak, due to ssM~-OsmT~, was used. This peak was not detected in the irradiated empty ampoule. The average amount (three results) was 0.44 ,ug* 0.03 J@ (’ 6.8%), i.e. 9.3 X 10m6 + 0.6 X 10-s (mol MO) 1-r. Most of the other peaks were also detected in the irradiated empty ampoule, particularly 122Sb 124Sb and 76As. It was observed that the areas per unit time of the corresponding photopeaks, corrected to the same decay time, were about the same as those measured in the spectrum of Fig. 3, which indicates that the protein solution does not contain ~t~ony and arsenic in measurable amounts. Iron and labile sulphide Non-haem iron(I1) was determined by the l,lO-phenanthroline method [S, 91. Labile sulphide was determined by an adaptation of the methods of Fogo and Popowski [lo] and Lovenberg et al. [9]. A high content of iron and labile sulphide per 120 000 mol. wt. was found (Table 1). Isoelectric point Analytical thin layer gel electrofocusing in polyacrylamide gel was used to estimate the isoelectric point on an LKB multiphor apparatus. A pH gradient between 2.5 and 6.0 was achieved using Ampholines. The low isoelectric point of 4.1 shows the acidic nature of the protein, although D. gigas (4Fe, 45) ferrodoxins are even more acidic. Amino acid composition Ammo acid analyses were carried out on a Beckman Multic~om amino acid analyser. Protein samples were hydrolysed in WC1(6 M) at 110 “C for 20 h, according to the method of Moore and Stein [ 11] . The values for threonine, serine and tyrosine were corrected for decomposition during hydrolysis. Cysteine and methionine were analysed after performic oxidation
Fig. 3. Gamma spectrum of irradiated quartz ampoule containing 0.5 ml protein aqueous solution, recorded 92 h after the end of 14 h irradiation. The counting time was 300 min and the intensity is shown as counts per channel on an arbitrary linear scale. The numbers on the spectrum are the energies (keV) of the photo peaks.
560 TABLE 2a Amino acid composition 7 4 4 12 7 5 12 5 14
LYS
His Arg Asp Thr Ser Glu Pro Gly
Ala CYS Val Met He Leu Tyr Phe Trp
13 3-4 8 3 5 9 4 4 ND
a 10-l (mol amino acid) (mol protein)-l. -JO5
1OL
07~~~
09-
08-
0.4
-
03,8
467
425
G m‘ g 06
-
g
322
5 v a 05 : e
T c
n
.$0.4
a J B
E -
-02
545
03
B 02
-
-
@l
c 0 I,-
7 I 300
I 400
I600
I 500 hlnml
Fig. 4. LTV-visible absorption spectra of (MO, Fe, S) protein from D. gigas: A, oxidised protein (0 - 0.5 optical density full scale); B, oxidised protein (0 - 1.0 optical density full scale); C, dithionite reduced protein (0 - 1.0 optical density full scale). as cysteic acid and methionine sulphone, respectively [ 12) . Results are given in Table 2. Amino acid composition is typical of (2Fe, 25) ferredoxins, with a high content in acidic residues and about two cysteine residues per iron atom.
N-terminal amino acid sequence Only a single type of N-terminal amino acid was found. The sequence near the N-terminal was determined on a Socosi PSlOO sequenator to be Gly-Leu-Gln-Lys-Val-(Leu)-(Thr)-Val-Asn-Gly 1 5
10
561
Optical spectra The optical absorption spectra of the protein in the oxidised and reduced states are shown in Fig. 4. The similarity of these spectra to those of other (ZFe, 2s) proteins suggests that the iron-sulphur centre is of this type. However the intensity of the absorption band at 467 nm in the oxidised protein is larger than usual so that there is an inversion in the A,,/ A 46, ratio when compared with typical (2Fe, 2s) proteins (0.98 is observed for the (MO, Fe, S) protein of D. gigas compared with 1.19 for Pseudomonas pu tida ferredoxin, for example [ 131. A similar inversion in the optical spectrum was observed in deflavo xanthine oxidase [l] . The presence of a (2Fe, 2s) centre was recently confirmed by EPR measurements [14]. Physiological role The purified (MO, Fe, S) protein was tested for activity in mediating electron transport between hydrogenase and sulphite reductase (desulphoviridin) [15]. No activity was detected. The formate dehydrogenase activity [16] was also tested; although the crude acidic protein fraction has this activity, it is absent from the purified (MO, Fe, S) protein [17]. The function of the protein therefore remains unknown.
Discussion A comparison of the spectroscopic data with those of (2Fe, 2s) ferredoxins [2] suggests that the cluster resembles that of “plant-type” ferredoxins. The existence of well-defined bands in the visible region of the spectra also indicates that the various iron-sulphur clusters of the protein are of the same type. There are two iron atoms and two labile sulphur atoms per 0.1 mol. wt., is of the order of magnitude of reported molecular weights of (2Fe, 2s) ferredoxins. It is interesting to note that there is a close similarity between the amino acid composition of the (MO, Fe, S) protein of D. gigas per 12 000 with those of plant type ferredoxins. A comparison of the N-terminal sequence of the (MO, Fe, S) protein with those of “plant-type” ferredoxins [ 181 shows similarities which may indicate that there is some symmetry in the polypeptide sequence : (MO, Fe, S) protein (D. gigas) Gly-LeuGln-Lys-Val-(Thr-LeubVal-Asn-Gly Ferredoxins Spinach Adrenal P. pu tida E. coli
Ala-Ala-Tyr-Lys-Val-Thr-Leu-Val-Thr-Pro-Thr Ser-Ser-Ser-Glu-Asp-Lys-Ile-Thr-Val-His-Phe Ser-Lys-Val-Val-Tyr-His-Asp Pro-Cys-Ile-Val-Ile-Leu-Tyr
562
Also the iron-sulphur-cysteine residues ratio per 0.1 mol. wt. is typical of ferredoxins. Other iron-sulphur compIex proteins ~on~~~g molybdenum have been reported [I, 2,161 but higher molecular weights have been estimated. Attempts to determine the physiological role have not been conclusive. Therefore this (MO, Fe, 8) protein from D. gigus is either of a new type or is a sub-unit of a more complex protein.
Acknowledgments We are indebted to Mrs. N. Forget and G. Bovier-Lapierre for their skilful technical help, to Dr. M. ~c~de~~ and R. Bourrelli (from the Laboratoire de Chimie Bacterienne) and to the Fermentation Plant of the University of Georgia for growing the bacteria. The authors wish to thank the Reactor Operation and Dosimetry Groups and Mrs. M. A. Gouveia of the L.F.E.N. for their assistance in performing the irradiations, flux monitoring and running the computer program.
References 1 R. C. Bray, in Paul D. Bayer (ed.), The Enzymes, Vol. XII, Academic Press, New York, 3rd e&s., 1975, p. 299. 2 D. 0 Hali, R. Cammack and K_ K. Rao, in A. Jacobs and M. Worwood (eds.), Iron in Biochemistry and Medicine, Academic Press, London, 1974, p. 279. 3 J. Le Gall, J. Mazza and N. Dragoni, Biochim. Biophys. Aeta, 99 (1965) 385. 4 M. Bruschi, E. C. Hatchikian, J. Le Gall, J. J. G. Moura and A. V. Xavier, Biochim. Biophys. Acta, in the press. 5 D. T. Summers, J, V. Maize1 and J. E. Darneil, Proc, Natl. Acad. Sci. U.S.A., 54 (1966) 505. 6 M. A. Gouveia and J. D, Cunha, L.F.E.N. Int. Rep., March, 1974. 7 J. Op de Beek, J. Radioanalyt. Chem., 11 (1972) 283. 8 A. E. Harvey, J. A. Smart, Jr. and E. S. Amis, Anal. Chem., 27 (1955) 26. 9 W. Lovenberg, B. B. Buchanan and J. 6. Rabinowitz, J. Biul. Chem., 238 (1963) 3899. 10 J. K. Fogo and M. Popowski, Anal. Chem., 21 (1949) 732. If S. Moore and M. H. Stein, in S. P. Colowiek and N. 0. Kaplan feds.), Methods in Enzymology, Vol. VI, Academic Press, New York, 1963, p. 819. 12 C. H. W. Him, in C. H, W. Him (ed.), Methods in Enzymology, Vol. XI, Academic Press, New York, 1967, p. 197. Proteins, 13 I. C. Gunsalus and J. D. Lipscomb, in W. Lovenberg (ed.), Iron-Sulphur Vol. 1, Academic Press, New York, 1973, p. 151. 14 J. J. 6. Moura, A, V. Xavier, M, Bruschi, J. Le Gall, D. 0. Hall and R. Carnmaek, Biochim. Biophys. Res. Commun,, in the press. 15 Jin PO-Lee and H. D. Peck, Jr., Biochem. Biophys. Res. Commun., 45 (1971) 583. 16 H. G. Enoch and R. L. Lester, J. Bioi. Chem., 250 (1975) 6693. 17 J. J. G. Moura, A. V. Xavier, M. Dubourdieu and J. Le Gall, unpublished data. 16 H. E. Knoell and J. Knappe, Eur. J. Biochem., 50 (1974) 246.
555
A MOLYBDENUM-CONT~~NG @Fe, 2s) PROTEIN FROM DESULPHOVIBRIO GIGAS, A SULPHATE REDUCER
J. J. G. MOURA and A. V. XAVIER Centro de Quimica Estruturat da Universidade de Lisboa, I.S.T., Lisbon-l {Portugal) M. BRUSC~I Labomtoire
and J. LEGALL de Chimie BactQritGzne, CNRS, 13274, Marseille, Cedex 2 (France)
J. M. P. CABRAL Labomtko
de Fisica e Engenharia Nucleares, Sacadm
(Portugal)
An acidic protein that contains molybdenum, iron and labile sulphide has been isolated from Desulphovibrio gigas, a sulphate reducer. The protein was purified by chromatographic procedures over DEAE-cellulose, Sephadex and alumina. The molecular weight, determined by polyacrylamide gel electrophoresis, is about 120 000. The amino acid composition is typical of the (ZFe, 2s) ferredoxins with a high propo~ion of acidic residues and about two cysteine residues per iron atom. It has twenty atoms of iron and twenty atoms of labile sulphide. One atom of molybdenum per molecule was determined by thermal neutron activation analysis. Optical absorption spectra indicate that the iron-sulphur chromophores are of the (2Fe, 2s) type.
Introduction Molybdenum has been shown to be present in different types of ironsulphur proteins. Examples of these complex iron-sulphur proteins are the molybdenum iron-sulphur flak hydroxylases (xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase and sulphite oxidase) which contain (2Fe, 2s) centres [ 11 and molybdenum-ferr~oxins where molybdenum is associated probably with (4Fe,4S) centres as sub-units of nitrogenase [2]. During the isolation of (4Fe,4S) ferredoxin from D. gigus we separated
*Presented at the Conference on “The Chemistry and Uses of Molybdenum”, University of Oxford, England, 31 August - 3 September, 1976, sponsored by Climax Molybdenum Co. Ltd. and the Chemical Society (Dalton Division).
556
R&we
mobilitv
Fig. 1. Electrophoretic polyacrylamide gel pattern (7.5% gel concentration) obtained on a single electrophoretic run. Seven proteins of known molecular weight were used: 1, cytochrome c, 12 460;2, ehymotrypsinogen A, 25 700; 3, egg_albumin, 46 000; 4 catalase, 60 000; 5, transferrin, 76 000; 6, Immunoglobulin G, 151000; 7, Immunoglobulm A, dim 360 000. References were run alone to confirm the assignments. Fig. 2. Plot of log (mol. wt.) (from data presented in Fig. 1) us. the electrophoretic mobility relative to bromophenol blue (polyacrylamide gel, polyacrylamide (7.5%), urea (3%), sodium dodecylsulphate (0.1%)).
another protein which proved to contain molybdenum and iron sulphur centres of the (2Fe,2S) type.
Experimental and results
Isolation of the (MO, Fe, S) protein
The isolation of this protein is based on its acidic characteristics. A frozen paste of cells (4 kg) was grown and treated as described previously [3]. All operations were carried at 0 - 4 “C unless otherwise stated. Tris-HCl and phosphate buffers (pH 7.6) of appropriate molarity were used. The fraction containing the (MO, Fe, S) protein was separated during the early stages of the purification of the three forms of the (4Fe, 45) ferredoxin [4]. The acidic extract [3] was adsorbed on a large DEAE-cellulose column and eluted with a discontinuous gradient 0.1 - 0.5 M Tris buffer. The fraction eluted with 0.2 - 0.3 M Tris buffer contains the (MO, Fe, S) protein together with flavoproteins, desulphoviridin and some cytochrome c3 (M.W., molecular
557
weight, 26 500) while the (4Fe, 4s) ferredoxin is eluted later (0.4 - 0.5 M Tris buffer). The (MO, Fe, S) protein was purified by rechromatography on DEAE-cellulose followed by columns of Sephadex G-50, silica gel and alumina. The purity of the protein was checked by polyacrylamide gel (Fig. 1) and has an absorbance ratio A2,s/A322 of 4.5. Molecular
weight
The molecular weight was estimated by polyacrylamide gel electrophoresis containing polyacrylamide (7.5%) in the presence of urea (3%) and sodium dodecylsulphate (0.1%) [ 51. The mobility of the (MO, Fe, S) protein was compared with seven proteins of known molecular weight used as standards and which gave the pattern shown in Fig. 1. A linear relationship between log (molecular weight) and the eiectrophoretic mobility referred to the dye (bromophenol blue) is obtained (see Fig. 2). A value of about 120 000 is estimated by this technique. The same value was obtained for the apoprotein which was produced after treatment with 3% HCl. No evidence of the presence of sub-units was found. Analysis of the constituents Molybdenum
of the protein
One atom of molybdenum per 120 000 mol. wt. was determined thermal neutron activation analysis.
by
Sample and standards. The sample was an aqueous solution of the (MO, Fe, S) protein (0.084 mM). Three aqueous standard solutions were prepared by dissolving NazMoO.+*2HzO p.a. in deionized water (2 X 10m5 M, 5 X 10M5 M and lo-*M). Aliquots (0.5 ml) of sample and standard solutions were introduced into about 5 ml quartz ampoules, cleaned beforehand in boiling HNO, and sealed at liquid nitrogen temperature under vacuum. Irradiation. The ampoules containing the sample and standards and an empty ampoule were irradiated for 14 h in the core grid of the RPI reactor (Sacavem) where the medium thermal neutron flux was 6.4 X 1012 n cmm2 s-l and the cadmium ratio (with cobalt) was 19.6. The set of ampoules was introduced into an aluminium can so that the ampoule containing the sample was symmetrically surrounded by the other four. Differences in neutron fluxes at the five positions at which the sample and standards stood were measured by means of 0.54% Co-Al alloy flux monitors. After allowing 24Na decay for 87 h, the irradiated substances were counted under exactly the same counting geometry. The counting time was between 2 and 5 h, depending on the concentration of molybdenum. Counting. The gamma-ray spectrometer consisted of a Ge( Li) coaxial detector (54 ml) connected through an Ortec model 120-4F preamplifier and an Ortec model 452 amplifier to an Inter-technique Didac 4000 multichannel analyser. This system had a resolution of about 2.5 keV for 1.333
558 TABLE 1 Characteristics of the D. gigas (MO, Fe, S) protein Molecular weight MO atoms/molecule Non-haem iron/molecule Labile sulphur/molecuIe Isoelectric point
120 000 1 - 20 - 20 4.1
MeV gamma rays. Calibration of the spectrometer was performed using standard gamma emitters. The spedrometric data were collected on punched tapes which were then processed in a PDP 15 computer using a computer program [6] which is an adap~tion of AKTAN [7]. The gamma spectrum of the irradiated ampoule containing the protein solution is shown in Fig. 3. For molybdenum determination, the 140.5 keV photopeak, due to ssM~-OsmT~, was used. This peak was not detected in the irradiated empty ampoule. The average amount (three results) was 0.44 ,ug* 0.03 J@ (’ 6.8%), i.e. 9.3 X 10m6 + 0.6 X 10-s (mol MO) 1-r. Most of the other peaks were also detected in the irradiated empty ampoule, particularly 122Sb 124Sb and 76As. It was observed that the areas per unit time of the corresponding photopeaks, corrected to the same decay time, were about the same as those measured in the spectrum of Fig. 3, which indicates that the protein solution does not contain ~t~ony and arsenic in measurable amounts. Iron and labile sulphide Non-haem iron(I1) was determined by the l,lO-phenanthroline method [S, 91. Labile sulphide was determined by an adaptation of the methods of Fogo and Popowski [lo] and Lovenberg et al. [9]. A high content of iron and labile sulphide per 120 000 mol. wt. was found (Table 1). Isoelectric point Analytical thin layer gel electrofocusing in polyacrylamide gel was used to estimate the isoelectric point on an LKB multiphor apparatus. A pH gradient between 2.5 and 6.0 was achieved using Ampholines. The low isoelectric point of 4.1 shows the acidic nature of the protein, although D. gigas (4Fe, 45) ferrodoxins are even more acidic. Amino acid composition Ammo acid analyses were carried out on a Beckman Multic~om amino acid analyser. Protein samples were hydrolysed in WC1(6 M) at 110 “C for 20 h, according to the method of Moore and Stein [ 11] . The values for threonine, serine and tyrosine were corrected for decomposition during hydrolysis. Cysteine and methionine were analysed after performic oxidation
Fig. 3. Gamma spectrum of irradiated quartz ampoule containing 0.5 ml protein aqueous solution, recorded 92 h after the end of 14 h irradiation. The counting time was 300 min and the intensity is shown as counts per channel on an arbitrary linear scale. The numbers on the spectrum are the energies (keV) of the photo peaks.
560 TABLE 2a Amino acid composition 7 4 4 12 7 5 12 5 14
LYS
His Arg Asp Thr Ser Glu Pro Gly
Ala CYS Val Met He Leu Tyr Phe Trp
13 3-4 8 3 5 9 4 4 ND
a 10-l (mol amino acid) (mol protein)-l. -JO5
1OL
07~~~
09-
08-
0.4
-
03,8
467
425
G m‘ g 06
-
g
322
5 v a 05 : e
T c
n
.$0.4
a J B
E -
-02
545
03
B 02
-
-
@l
c 0 I,-
7 I 300
I 400
I600
I 500 hlnml
Fig. 4. LTV-visible absorption spectra of (MO, Fe, S) protein from D. gigas: A, oxidised protein (0 - 0.5 optical density full scale); B, oxidised protein (0 - 1.0 optical density full scale); C, dithionite reduced protein (0 - 1.0 optical density full scale). as cysteic acid and methionine sulphone, respectively [ 12) . Results are given in Table 2. Amino acid composition is typical of (2Fe, 25) ferredoxins, with a high content in acidic residues and about two cysteine residues per iron atom.
N-terminal amino acid sequence Only a single type of N-terminal amino acid was found. The sequence near the N-terminal was determined on a Socosi PSlOO sequenator to be Gly-Leu-Gln-Lys-Val-(Leu)-(Thr)-Val-Asn-Gly 1 5
10
561
Optical spectra The optical absorption spectra of the protein in the oxidised and reduced states are shown in Fig. 4. The similarity of these spectra to those of other (ZFe, 2s) proteins suggests that the iron-sulphur centre is of this type. However the intensity of the absorption band at 467 nm in the oxidised protein is larger than usual so that there is an inversion in the A,,/ A 46, ratio when compared with typical (2Fe, 2s) proteins (0.98 is observed for the (MO, Fe, S) protein of D. gigas compared with 1.19 for Pseudomonas pu tida ferredoxin, for example [ 131. A similar inversion in the optical spectrum was observed in deflavo xanthine oxidase [l] . The presence of a (2Fe, 2s) centre was recently confirmed by EPR measurements [14]. Physiological role The purified (MO, Fe, S) protein was tested for activity in mediating electron transport between hydrogenase and sulphite reductase (desulphoviridin) [15]. No activity was detected. The formate dehydrogenase activity [16] was also tested; although the crude acidic protein fraction has this activity, it is absent from the purified (MO, Fe, S) protein [17]. The function of the protein therefore remains unknown.
Discussion A comparison of the spectroscopic data with those of (2Fe, 2s) ferredoxins [2] suggests that the cluster resembles that of “plant-type” ferredoxins. The existence of well-defined bands in the visible region of the spectra also indicates that the various iron-sulphur clusters of the protein are of the same type. There are two iron atoms and two labile sulphur atoms per 0.1 mol. wt., is of the order of magnitude of reported molecular weights of (2Fe, 2s) ferredoxins. It is interesting to note that there is a close similarity between the amino acid composition of the (MO, Fe, S) protein of D. gigas per 12 000 with those of plant type ferredoxins. A comparison of the N-terminal sequence of the (MO, Fe, S) protein with those of “plant-type” ferredoxins [ 181 shows similarities which may indicate that there is some symmetry in the polypeptide sequence : (MO, Fe, S) protein (D. gigas) Gly-LeuGln-Lys-Val-(Thr-LeubVal-Asn-Gly Ferredoxins Spinach Adrenal P. pu tida E. coli
Ala-Ala-Tyr-Lys-Val-Thr-Leu-Val-Thr-Pro-Thr Ser-Ser-Ser-Glu-Asp-Lys-Ile-Thr-Val-His-Phe Ser-Lys-Val-Val-Tyr-His-Asp Pro-Cys-Ile-Val-Ile-Leu-Tyr
562
Also the iron-sulphur-cysteine residues ratio per 0.1 mol. wt. is typical of ferredoxins. Other iron-sulphur compIex proteins ~on~~~g molybdenum have been reported [I, 2,161 but higher molecular weights have been estimated. Attempts to determine the physiological role have not been conclusive. Therefore this (MO, Fe, 8) protein from D. gigus is either of a new type or is a sub-unit of a more complex protein.
Acknowledgments We are indebted to Mrs. N. Forget and G. Bovier-Lapierre for their skilful technical help, to Dr. M. ~c~de~~ and R. Bourrelli (from the Laboratoire de Chimie Bacterienne) and to the Fermentation Plant of the University of Georgia for growing the bacteria. The authors wish to thank the Reactor Operation and Dosimetry Groups and Mrs. M. A. Gouveia of the L.F.E.N. for their assistance in performing the irradiations, flux monitoring and running the computer program.
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