FEMS Microbiology Letters 130 (1995) 193-200
Biochemical and EPR characterization of a high potential
iron-sulfur protein in Thiobacillus ferrooxidans
Christine Cavazza, Bruno Guigliarelli, Patrick Bertrand, Mireille Bruschi
Biobergt?tique
et Ingtkierie
*
des Protkines, CNRS, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France
Received 20 March 1995; revised 22 May 1995; accepted25 May 1995
Abstract
A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobucilhs ferrooxiduns using the
periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of
5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15
residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences
were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified
protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized
state was attributed to a [4Fe-4S13+ cluster and the redox potential has been determined to be +380 mV.
Keywords:
High potential iron-sulfur protein; iro gene; Thiobacillus ferrooxidans
1. Introduction
Microbial leaching is an industrial process which
is used for the acid draining of metal sulfide ore
mines [l] and Thiobacillus ferrooxidms is the main
bacterium involved in this process [2]. This organism
is a chemolithotrophic
aerobic bacterium which is
able to oxidize iron and reduced inorganic sulfur
compounds in acidic conditions. Iron oxidation is of
considerable importance in bioleaching processes because ferric iron oxidizes sulfide minerals and conse-
quently enables the solubilisation of metals and the
release of inorganic sulfur species [3]. The electron
transfer pathway from ferrous iron (which is the
* Corresponding author. Tel.: +33 91 16 41 44; Fax: +33 91
77 95 17
primary energy-generating process of the bacterium)
to molecular oxygen includes several redox components which are either located in the periplasmic
space or associated with the internal membrane as in
the case of the cytochrome oxidase. Fe2+ is a substrate with a high redox potential which therefore
constitutes a particularly weak reducing agent (I$, =
+780 mV with the Fe2+/Fe3+ couple). Little energy is consequently
released from the electron
transfer chain and its components exhibit very high
redox potentials. Various metalloproteins
have been
isolated from T. ferrooxiduns: several cytochromes c
and b, a cytochrome a,-type oxidase and a’ blue
copper protein (msticyanin) [4]. Rusticyanin, which
is the only redox protein in this bacterium present in
large amounts (up to 5% of the total cell proteins)
[5], has been fully characterized [6-g]. Moreover, the
presence of an iron-sulfur protein has been reported
0378-1097/95/$09.50 8 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 0378-1097(95)00205-7
194
C. Cauaua et al. / FEMS Microbiology Letters 130 (19951 193-200
in T. ferrooxidans. Fry et al. [lo] have observed that
whole cells, after acid washing in cold HCl (pH 2.5)
exhibited an EPR signal in the oxidized state at
g = 2.005 and suggested this signal might be attributable to a [3Fe-xS] cluster belonging to a membrane-bound component of the Fe*+ oxidoreductase
enzyme responsible for the direct oxidation of Fe2+
to Fe3+. Fukumori et al. [11,12] subsequently purified the Fe(B) cytochrome css2 oxidoreductase,
a
soluble iron-sulfur protein with a molecular mass of
63 kDa, containing 18-20 atoms of non-heme iron
and 6 atoms of inorganic sulfide. Rusticyanin was
not reduced by this enzyme while cytochrome cs5*,
in the presence of FeSO, at pH 3.5, was rapidly
reduced. In 1992, Kusano et al. [13] cloned and
sequenced the iro gene, encoding this enzyme. The
results of sequence analysis and comparisons with
other iron-sulfur
proteins sequences have suggested
that the Iro enzyme is a high redox potential ferredoxin consisting of several 5884-Da subunits, each
containing one [4Fe-4S] cluster. However, Blake and
Shute [14] have noted the existence of several
anomalies as regards the hypothesized catalytic function of this iron-sulfur
protein.
In the present study, we isolated a soluble ironsulfur protein
using the periplasmic
extraction
method. With this method, we obtained a sufficient
large amount of highly purified protein to be able to
determine some of its biochemical and biophysical
characteristics and the nature of the iron-sulfur cluster. The data obtained on this protein were compared
with the corresponding data on the iro gene.
2.2. Release of periplasmic proteins
In order to isolate the periplasmic proteins, cells
(100 g) were suspended in 200 ml of 50 mM Tris .
HCl and 50 mM EDTA (pH 9.01, and stirred for 30
min at 37°C as described by Van der Westen et al.
[16]. The cells were then removed from the suspension by centrifugation (20 min at 10 000 X g) and the
supernatant containing the periplasmic proteins was
adjusted to pH 6.0 (using 1 M phosphate buffer) and
dialysed.
2.3. Optical absorption spectra
The visible and ultraviolet absorption spectra of
the protein were determined with a Beckman DU
7000 spectrophotometer.
Molar extinction
coefficients at the absorption maxima were determined
relative to the protein concentration
determined by
performing amino acid analysis.
2.4. Isoelectric point measurements
The isoelectric point of the protein was determined by performing isoelectric focusing using a
Phast System
apparatus
from Pharmacia
LKB
Biotechnology [17]. Phast Gel IEF 3-9, which covers the pH range 3-9, and ampholine polyacrylamide
gel plates from Pharmacia (pH range 3.5-9.5) were
used together with a Pharmacia broad-range pl calibration kit containing proteins with different isoelectric points ranging from 3 to 10.
2. Materials and methods
2.1. Microorganism and culture procedure
Thiobacillus ferrooxidans was kindly supplied by
Dr. D. Morin (Bureau des Recherches Ghologiques
et Mini&es, Orltans, France) This bacterium has
been isolated from drainage water at the Salsigne
sulfur mine (France). It was grown at pH 1.6 in 9 K
Silverman
and Lundgreen
medium [15] supplemented with CuSO, .5H,O at a concentration of 1.6
mM. Large-scale cultivation of the organism was
performed in 300 1 of the above medium with a
home-made polyprene fermenter.
2.5. Molecular mass determination
The molecular mass of the protein was determined by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions on a Pharmacia PhastSystem with PhastGel
12% polyacrylamide and PhastGel SDS buffer strips.
Mass spectrometry analyses were carried out on a
Perkin-Elmer
Sciex API III triple quadripole mass
spectrometer equipped with a nebulizer-assisted
electrospray (ionspray) source. The spectra were recorded
as described previously [18].
C. Cauana et al./ FEMS MicrobiologyLetters130 (1995) 193-200
2.6. Redox titration and EPR spectroscopy
The titration was carried out at 21°C in a 50 mM
ammonium acetate (pH 4.8) solution kept in an
argon atmosphere. The protein concentration was 25
PM. The solution was initially reduced with sodium
dithionite before being progressively oxidized with
small amounts of a concentrated solution of potassium ferricyanide. At potentials more positive than
480 mV, potassium hexachloroiridate (IV) was used
as an oxidant. The solution potentials were measured
with a combined Pt-Ag/AgCl KC1 (3 M) electrode.
In the text, the potentials are given with respect to
the standard hydrogen electrode. At each equilibrium, a sample was drawn and anaerobically transferred into calibrated EPR tubes which were rapidly
frozen. EPR experiments and spin quantitations were
carried out as described previously [19].
2.7. Amino acid analysis and protein sequencing
For the amino acid analysis, protein samples were
hydrolysed in 200 ml of 6 M HCl in sealed vacuum
tubes at 110°C for 24 and 72 h and then analysed
with a Beckman amino acid analyzer (System 6300).
Sequence determinations were carried out with an
Applied Biosystems A470 gas-phase sequenator.
Quantitative determinations were performed on the
phenyl thiohydantoin derivatives by means of highpressure liquid chromatography (Waters Associates,
Inc.) monitored by a data and chromatography control station (Waters 840).
S-carboxymethylated protein was prepared by dissolving the protein in 0.5 M Tris * HCl (pH 9.01, 8 M
urea and 20 mM EDTA, and treating it with
iodoacetic acid, as described by Crestfield et al. [20].
Methionine peptides were produced by reacting carboxymethylated protein in 70% trifluoroacetic acid
with a 300-fold excess of CNBr for 24 h at room
temperature in the dark.
3. Results
3.1. Purification of the iron-suljbr protein
The periplasmic fraction was dialysed for 4 h
versus 10 mM phosphate buffer (pH 6.0). The T.
195
ferrooxidans iron-sulfur protein was purified in three
steps. The dialysed solution was applied to a Bio Gel
hydroxyapatite column (3 X 7 cm) (Bio-Rad) equilibrated with 3Ci mM sodium phosphate buffer (pH
6.0) in order to adsorb the rusticyanin and the cytochrome fraction. After being diluted (X 1.51, the
non-adsorbed fraction was subjected to a carboxymethylcellulose column (2.5 X 7 cm) (Whatman) equilibrated with 20 mM sodium phosphate
buffer (pH 6.0). The brownish-green band containing
reduced iron-sulfur protein was eluted with 100 mM
buffer and concentrated using centricon. The resulting fraction was applied to a monoS HR S/5 column
(Pharmacia Fine Chemicals) equilibrated with 50
mM ammonium acetate (pH 4.8). The proteins were
eluted with a 50 mM to 1 M gradient. The iron-sulfur
protein was eluted with 100 mM buffer and was
found to be pure by SDS-PAGE. 1 mg of pure
protein was obtained from 100 g of bacteria (wet
weight). This protein is basic (pl = 9) and stable in
the pH range 2-9 (data not shown). It can be stored
for several months in a 10 mM (HCl, KCl) buffer
(pH 2.1) at -20°C.
3.2. Molecular mass
The apparent molecular mass deduced from the
SDS-PAGE analysis was about 24000 Da with the
native form and 6000 Da when the sample was
boiled for 1 min. The band corresponding to about
24000 Da was diffuse, while the 6000-Da band was
clear-cut (data not shown). The mass spectra showed
two peaks at 5532 Da (corresponding to the apoprotein) and 5882 Da (including the cluster Fe-S). These
results suggest that the native form consists of four
identical subunits.
3.3. Optical spectra
The optical spectra showed a characteristic peak
at 388 nm which is typical of proteins containing
iron-sulfur clusters (Fig. 1). The highest 388 mn to
280 nm absorbance ratio obtained was 0.59 in the
case of the brownish-green reduced HiPIP. The extinction coefficient of reduced protein at 388 mn was
about 65.7 mM_’ cm-’ with the tetrameric form,
which is consistent with the presence of one iron-
C. Cauazza et al. / FEMS Microbiology Letters 130 (1995) 193-200
196
0.25
-
I
01
300
504
400
WAVELENGHT
sulfur
cluster
per
subunit
I
I
I
I
310
320
330
340
(nm)
Magnelic
Fig. 1. Absorption spectra of T. ferrooxidans HiPIP. The protein
(25 PM) was dissolved in 50 mM ammonium acetate buffer (pH
4.8). The oxidized form (with K,IrCI,,
120 PM) is indicated by
the solid line, and the reduced form (with sodium dithionite, 1.5
mM) by the dotted line.
showed a purplish-brown
I
[21]. The oxidized
color.
form
3.4. Iron content
The iron content, as determined by performing
plasma emission spectroscopy (using a Jobin Yvon
model JY 38 apparatus), was 16.5 atoms of iron per
molecule, i.e. 4.1 atoms of iron per subunit. This
result suggests the presence of one cluster [4Fe-4S]
per subunit.
Fteld /ml
Fig. 2. EPR spectrum of the fully oxidized Thiobacillus ferrooxidam iron-sulfur
protein. Experimental conditions: temperature,
15 K; microwave frequency, 9.333 GHz; microwave power, 0.4
mW; modulation frequency, 100 kHz; modulation amplitude, 0.5
mT.
of which the two centers are coupled by a cooperative redox interaction equal to + 100 mV [19] (Fig.
3).
3.6. Amino acid analysis and N-terminal
determination of the subunit
sequence
The amino acid composition of the protein was
determined by performing amino acid analysis on the
basis of the molecular mass of the subunit (5532
3.5. EPR experiments
No EPR signal was observed with samples poised
at potentials less positive than 350 mV. During the
oxidation, an axial EPR signal characterized by g,,
= 2.127, g, = 2.034 gradually appeared. The shape
of this signal did not change during the titration, and
its amplitude was fully developed at 500 mV, giving
an integrated intensity corresponding
to 4.2 + 0.2
spin per molecule (Fig. 2). The variations in the
signal amplitude as a function of the redox potential
are given in Fig. 3. These variations did not obey a
Nemst curve with n = 1. A good fit was obtained by
assuming the four iron-sulfur
centers of the tetramer
to be characterized
by E, = 380 mV and to be
arranged in two independent pairs of centers in each
ElmV
300
400
500
600
Fig. 3. Redox titration of the HiPIP EPR signal. The peak-to-peak
amplitude of the g = 2.034 line was measured as a function of the
redox potential on EPR spectra recorded as in Fig. 1. The dashed
line is a Nemst curve with n = 1 and E” = + 430 mV. The solid
line is the best fit obtained taking two equivalent redox centers
with microscopic redox potentials e, = e, = +380 mV, coupled
by a cooperative redox interaction I,, = + 100 mV.
C. Cauazza et al. / FEMS Microbiology Letters 130 (I 995) 193-200
Da). This analysis showed the presence of four
cysteines per subunit, which is in agreement with the
fact that this protein contains a [4Fe-4S] cluster,
ligated by four cysteines. Upon comparing the amino
acid composition deduced from the nucleotide sequence of the iron-sulfur protein isolated from another strain of T. ferrooxidans (Iro protein) [13] with
the amino acid composition obtained in our study,
the two compositions showed only two differences
(7 Ala instead of 6 and 5 Lys instead of 4).
Automated Edman degradation of 1 nmol of the
carboxymethylated protein yielded the amino-terminal sequence up to the 14th cycle. The N-terminal
amino acid sequence (Gly-Ser-Met-Pro-Lys-Ala-AlaVal-Gln-Tyr-Gln-Asp-Thr-Pro) was identical to that
derived from the nucleotide sequence of the iro gene
1131.
4.Discussion
In this study, in which the periplasmic extraction
method was used for the first time, we established
that the purified iron-sulfur protein is an acid-stable
periplasmic protein. This method has turned out to
be faster, and thus less denaturing than performing
protein purification on cells treated with a French
press. This protein is a tetramer, while Fukumori et
al. [ll] have purified a multimeric 63-kDa form
(corresponding to the presence of 10 subunits) with
18-20 atoms of non-heme iron and 6 atoms of
inorganic sulfide as compared with 4 Fe and 4 S in
the case of a 6000-Da subunit. In our study, we did
not isolate the 63-kDa form throughout the purification procedure and the iron content was in agreement
with the presence of four subunits, each containing
one [4Fe-4S] cluster. Moreover, the integrity of the
[4Fe-4S] clusters was demonstrated by the EPR study.
The EPR spectrum given by the oxidized form of the
iron-sulfur protein from T. ferrooxiduns, which is
characterized by an axial signal with g,,= 2.127,
g I = 2.034, is very similar to that given by the
high-potential iron-sulfur protein of Chromatium
vinosum [22,23]. This spectrum can therefore be
attributed to a [4Fe4S13+ cluster, the extra feature at
g = 2.074, indicating that the cluster is probably
present under different electronic states. The intensity measurements confirmed the presence of one
197
[4Fe-4S13+/‘+ cluster per subunit. Since the [4Fe4S13+12+ redox equilibrium is a one-electron process, the divergence of the data given in Fig. 3 from
a simple n = 1 Nemst curve indicates that the four
centers present in a molecule are coupled by redox
interactions. The simplest model describing this situation is that leading to the theoretical curve given in
Fig. 3, but other models involving several interaction
potentials could obviously be used to fit the data.
The occurrence of cooperative interactions in the
tetrameric form might help the protein to accept
electrons at high potential. A less complex redox
behaviour was observed in the case of the protein
from C. uinosum, which is a monomeric protein.
However, this protein dimerizes in the presence of a
high concentration of NaCl, so that a complex EPR
spectrum arising from the magnetic interactions between pairs of [4Fe-4S13+ clusters develops 1241.No
such spectrum is observed in the case of the tetrameric protein from T. ferrooxidms. Moreover, all
the HiPIP characterized so far are monomers, except
for iso- HiPIP from the halophilic purple photosynthetic bacterium Rhodospirillum salinarum, which is
also a tetramer [25].
The amino acid composition and N-terminal sequence of the HiPIP both show considerable similarity with the amino acid sequence deduced from the
iro gene [13]. The iro gene was cloned, using degenerate oligonucleotides based on the N-terminal sequence of the Fe(R) cytochrome c552 oxidoreductase
(previously characterized by Fukumori et al. [ll]>
electroblotted onto PVDF membranes. Upon carrying out a computer search on protein data bases, the
Iro protein was found to have a high degree of
homology with high redox potential iron-sulfur proteins (HiPIP). The presence of a signal sequence has
suggested that the Iro enzyme might be a periplasmic
protein, while Ehrlich et al. [26] postulated that the
iron-sulfur protein might be cytoplasmic. This enzyme has been described as a Fe(R)-oxidizing enzyme catalysing the oxidation of Fe”’ ions with
cytochrome css2 acting as the electron acceptor [ll].
Ehrlich et al. [26], however, have interpreted these
data as indicating that the 63000-Da iron-sulfur
protein might be a denatured form which has lost a
number of [4Fe-4S] clusters and that a transient
intermediate of this protein might trigger the autooxidation of Fe(R) along with the concomitant reduc-
198
C. Cauazza et al. / FEMS Microbiology Letters 130 (1995) 193-200
tion of the cytochrome. Moreover, Blake and Shute
[14] have partially purified an iron:rusticyanin
oxidoreductase which is thought to be the primary
cellular oxidant of ferrous ions in the iron respiratory
electron transport chain of T. ferrooxidans instead of
the HiPIP. Therefore, the exact role of the HiPIP is
still largely misunderstood and it will be necessary to
carry out further studies to determine the functional
meaning of this protein.
Acknowledgements
The authors gratefully acknowledge the Fermentation Plant Unit (LCB, Marseille, France) for growing
the bacteria, and Nicole Zylber and Jacques Bonicel
(Protein Sequencing Unit, Marseille, France) for performing amino acid analysis and N-terminal amino
acid sequencing.
We thank Dr. Franqoise Guerlesquin (BIP, Marseille, France) for helpful discussions, Dr. Jean-Claude Germanique for the iron content analysis and Eric Forest (IBS, Grenoble, France)
for mass spectroscopy analysis. C.C. acknowledges
the support of a graduate Scholarship from 1’Agence
de 1’Environnement
et de la Maitrise de 1’Energie
(ADEME). This study was supported by a grant from
1’Agence de 1’Environnement
et de la Maitrise de
1’Energie (ADEME),
le Bureau des Recherches
Geologiques
et Mini&es (BRGM) and COGEMA
(France).
References
[l] Brock, T.D. and Madigan, M.T. (1991) Biology of Microorganisms, 6th edn. Prentice-Hall, New York, NY.
[2] Ewart, D.K. and Hughes, N.H. (1991) The extraction of
metals from ores using bacteria. Adv. Inorg. Chem. 36,
103-135.
[3] Lundgren, D.G. and Silver, M. (1980) Ore leaching by
bacteria. Ann. Rev. Microbial. 34, 263-283.
[4] Ingledew, W.J. (1982) Thiobacillus ferrooxidans. The bioenergetics of an acidophilic chemolithotroph.
Biochim. Biophys. Acta 683, 89-117.
[S] Cox, J.C. and Boxer, D.H. (19781 The purification and some
properties of rusticyanin, a blue copper protein involved in
the iron(H) oxidation
from Thiobacillus ferrooxidans.
B&hem. J. 174, 497-502.
[6] Cox, J.C., Aasa, R. and Malmstrom,
B.G. (1978) EPR
studies on the blue copper protein rusticyanin. A protein
involved in Fe*+ oxidation at pH 2.0 in Thiobacillus ferrooxidans. FEBS L&t. 93, 157-160.
[7] Nunzi, F., Woudstra, M., Camp&se, D., Bonicel, J., Morin,
D. and Bruschi, M. (1993) Amino-acid sequence of rusticyanin from Thiobacillus ferrooxidans and its comparison
with other blue copper proteins. Biochim. Biophys. Acta
1162, 28-34.
[81Nunzi, F., Haladjian, J., Bianco, P. and Bruschi, M.(1993)
Electron-transfer reaction of rusticyanin, a ‘blue’-copper protein from Thiobacillus ferrooxidans, at modified gold electrodes. J. Electroanal. Chem. 352, 329-335.
[91 Hunt, A.H., Toy-Palmer, A., Assa-Mint, N., Cavanagh, J.,
Blake, R.C. II and Dyson, H.J. (1994) Nuclear magnetic
resonance “N and ‘H resonance assignments and global fold
of rusticyanin. J. Mol. Biol. 244, 370-384.
1101Fry, IV., Lazaroff, N. and Packer, L. (1986) Sulfate-dependent iron oxidation by ThiobaciNw ferrooxidans: characterization of a new EPR detectable electron transport component
on the reducing side of rusticyanin. Arch. Biochem. Biophys.
246, 650-654.
1111Fukumori, Y., Yano, T., Sato, A. and Yamanaka, T.(1988)
Fe(B)-oxidizing enzyme purified from Thiobacillus ferrooxidam. FEMS Microbial. Lett. 50, 169-172.
[I21 Yamanaka, T., Fukumori, Y., Yano, T., Kai, M. and Sato, A.
(1991) Enzymatic mechanisms in the ‘dehydrogenation’
of
ferrous ions by Thiobacillus ferrooxidans. Dev. Geochem. 6,
267-273.
[I31 Kusano, T., Takeshima, T., Sugawara, K., Inoue, C., Shiratori, T., Yano, T., Fukumori, Y. and Yamanaka, T. (1992)
Molecular cloning of the gene encoding Thiobacillus ferrooxidans Fe(E) oxidase. J. Biol. Chem. 267, 11242-11247.
[141 Blake, R.C. II and Shute, E. (1994) Respiratory enzymes of
Thiobacillus ferrooxidans. Kinetic properties of an acid-stable iron: rusticyanin oxidoreductase. Biochemistry 33, 92209228.
D51 Silverman, M.P. and Lundgren, D.G. (1959) Studies on the
chemoautotrophic
iron bacterium T. ferrooxidans. I. An improved medium and a harvesting procedure for securing high
cell yield. J. Bacterial. 77, 642-647.
[16] Van der Westen, H.M., Mayhew, S.G. and Veeger, C. (1978)
Separation of hydrogenase from intact cells from Desulfouibrio vulgaris. FEBS Lett. 86, 122-126.
[17] Haff, L.A., Fagerstam, L.A. and Barry, A.R. (1983) Use of
electrophoretic
titration curves for predicting optimal chromatograpbic conditions for fast ion-exchange chromatography of protein. J. Chromatogr. 266, 409-425.
[18] Dolla, A., Florens, L., Bianco, P., Haladjian, J., Voordouw,
G., Forest, E., Wall, J., Guerlesquin, F. and Bruschi, M.
(1994) Characterization
and oxidoreduction properties of cytochrome ca after heme axial ligand replacements. J. Biol.
Chem. 269, 6340-6346.
[19] Guigliarelli, B., Asso, M., More, C., Augier, V., Blasco, F.,
Pommier, J., Giordano, G. and Bertrand, P. (1992) EPR and
redox characterization
of iron-sulfur centers in nitrate reductases A and 2 from Escherichia coli. Eur. J. Biochem. 207,
61-68.
[20] Crestfield, A.M., Moore, S. and Stein, W.H. (1963) The
C. Cavazza et al. /FEMS
Microbiology Letters 130 (1995) 193-200
preparation and enzymatic hydrolysis of reduced and Scarboxymethylated proteins. J. Biol. Chem. 238, 622-627.
1211Hong and Rabinowitx (1970) Molar extinction coefftcient
and iron and sulfide content of clostridial ferredoxin. J. Biol.
Chem. 245,4982-4987.
[22] Antanaitis, B.C. and Moss, T.H. (1975) Magnetic studies of
the four-iron high-potential, non-heme protein from Chromatium vinosum. Biochim. Biophys. Acta 405, 262-279.
[23] Moulis, J.M., Lutz, M., Gaillard, J. and Noodleman, L.
(1988) Characterization of [4Fe-Se]2*/3+ high potential
iron-sulfur protein from Chromatium vinosum. Biochemistry
27, 8712-8719.
[24] Dunham, W.R., Hagen, W.R., Fee, J.A., Sands, R.H., Dun-
199
bar, J.B. and Humblet, C. (1991) An investigation of Chromatium uinosum high potential iron-sulfur protein by EPR
and Mossbauer spectroscopy: evidence for a freexing-induced dimerixation in NaCl solutions. Biochim. Biophys.
Acta 1079, 253-262.
[z] Meyer, T.E., Fitch, J., Bar&h, R.G., Tollin, D. and Cusanovitch, M.A. (1990) Unusual high redox potential ferredoxins and soluble cytochromes from the moderately
halophilic purple phototrophic bacterium Rhodospirillum
safinarum. B&him. Biophys. Acta 1017, 118-124.
[26] Ehrlich, H.L., Ingledew, W.J. and Salerno, J.C. (1991) In:
Variations in Autotrophic Life (Shively, J.M. and Barton,
L.L., Eds.), pp. 147-170. Academic Press, London.