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FEMS Microbiology Letters 121 (1994) 165-170
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
165
FEMSLE 06109
Properties of a thermostable 4Fe-ferredoxin
from the hyperthermophilic bacterium
Thermotoga maritima
Jenny M. Blamey, Swarnalatha M u k u n d and Michael W.W. A d a m s *
Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
(Received 5 May 1994; revision received 7 June 1994; accepted 8 June 1994)
Abstract: A ferredoxin has been purified from one of the most ancient and most thermophilic bacteria known, Thermotoga
maritima, which grows up to 90°C. The reduced protein (M r approx. 6300) contains a single S = 1/2 [4Fe-4S] 1÷ cluster with
complete cysteinyl ligation, and was unaffected after incubation at 95°C for 12 h. It functioned as an electron carrier for T.
maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein
are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and
moderately thermophilic bacteria.
Key words: Thermotoga maritima; Fermentation; Thermostability; Hyperthermophilic Archaea; Evolution
Introduction
Thermotoga maritima [1] represents one of the
two most thermophilic and most ancient genera
within the bacterial domain [2], and is usually
referred to as a hyperthermophile. It grows up to
90°C by the fermentation of carbohydrates with
lactate, acetate, CO 2 and H 2 as the products. If
elemental sulfur (S°) is also added to the growth
medium, the organism reduces it to H2S [1]. T.
maritima therefore resembles many of the hyperthermophilic archaea such as species of Pyrococcus and Thermococcus [3]. These organisms also
* Corresponding author. Tel.: (706) 5422060; Fax: (706) 542
0229.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 2 5 9 - T
grow at 90°C, have S%reducing, saccharolytic
metabolisms, and are the most slowly evolving
organisms within the their domain [2,3]. T. maritima differs from the archaea, however, in that it
couples glucose oxidation to H 2 production via a
conventional Embden-Meyerhof-Parnas pathway,
pyruvate ferredoxin oxidoreductase [4] and an
Fe-hydrogenase [5]. In contrast, hyperthermophilic archaea are proposed to contain an unusual type of ferredoxin-dependent EntnerDoudoroff pathway [6], a novel type of pyruvate
ferredoxin oxidoreductase (see [41) and a Ni-hydrogenase [7]. The ferredoxins from the archaea
P. furiosus and T. litoralis are extremely thermostable and contain a single 4Fe-cluster [8,9]. In
contrast, mesophilic fermentative bacteria typically contain thermally labile 8Fe-type ferredox-
166
ins. Herein we report the purification and characterization of the ferredoxin from the hyperthermophilic bacterium, T. maritima.
Materials and Methods
Growth of the bacterium and purification of ferredoxin
Thermotoga maritima (DSM 3109) was grown
as previously described [5]. The ferredoxin was
routinely purified from 200 g of cells (wet weight)
at 23°C under anaerobic conditions [5]. The procedure was the same as for the purification of T.
maritima hydrogenase, up to and including the
first Q Sepharose (Pharmacia LKB) column [5].
The ferredoxin started to elute when 0.38 M
NaC1 was applied to the column. Fractions (100
ml) with an m390/A280 ratio greater than 0.13
(600 ml) were then applied to a column (5 x 30
cm) of hydroxyapatite (Behring) previously equilibrated with 50 mM Tris. HCI (pH 8.5). A gradient from 0 to 0.3 M potassium phosphate (1.5 I)
in the same buffer was applied at 2.5 ml rain -1
and the ferredoxin started to elute as soon as the
gradient began. Fractions (20 ml) with a
A39o/A28 o ratio above 0.28 were combined (160
ml) and concentrated to approximately 20 ml
using a column (HR 10/10) of Q Sepharose
equilibrated with 50 mM Tris (pH 8.0). The sample was applied directly to the column and eluted
at 0.5 ml min-~ in 3-ml fractions with a gradient
(40 ml) from 0 to 1 M NaC1. The concentrated
ferredoxin was applied to a column (6.0 × .60 cm)
of Superdex 75 (Pharmacia LKB) equilibrated
with buffer containing 0.2 M NaC1. Fractions (15
ml) with a A390/Azso value above 0.70 were combined (60 ml, 80 rag) and concentrated using a Q
Sepharose column. The ferredoxin was stored as
pellets in liquid N 2.
used as standards. SDS-electrophoresis, analyses
for iron, acid-labile sulfide, tryptophan and cysteine, and the preparation of reduced apoferredoxin were all carried out as previously described
[8]. The concentrations of samples of pure ferredoxin were estimated by a heat-modified version
of the Lowry method (see [8]). The results agreed
(_+ 8%) with the amounts of protein recovered by
quantitative recovery of amino acids (see [9]).
E P R spectroscopy [4] and the assays of hydrogenase [9] and of pyruvate ferredoxin oxidoreductase [4] of T. maritima were carried out as indicated.
Results and Discussion
Molecular properties
Purified T. maritima ferredoxin gave rise to a
single protein band after SDS-electrophoresis
(using 20% (w/v) acrylamide) and eluted as a
single peak from a gel filtration column. The M r
value obtained using both techniques was 7300
+ 1500, indicating that it is a monomeric protein.
The UV-visible absorption spectra of the airoxidized and dithionite-reduced forms (for exampie, see Fig. 1) were typical of this class of protein
(see [10]). The m39o/A280 ratio was 16.3 mM -1
c m - ' and the molar absorbance at 390 nm was
0.74. The protein contained 4.2 +_ 0.6 and 3.9 + 1.2
g atoms (mol i r o n ) - ' and acid-labile sulfide, respectively, suggesting the presence of a single
4Fe-center. The amino acid composition of the
ferredoxin was (in residues mol-1): B, 8; T, 2; S,
2; Z, 7; P, 4; G, 6: A, 7; C, 6; V, 5; M,1; I, 2; L, 4;
F, 2; K, 4; R, 1 (from four separate determinations), for a total of 61 residues. Tyrosine, histidine and tryptophan were not detected. From
these data, the ferredoxin has a M r value of 6331
(including the iron and sulfide), comparable to
that of P. furiosus ferredoxin [9].
Analytical methods'
The molecular mass was estimated using a
column (16/30) of Superdex 75 using 50 mM
Tris. HC! (pH 8.0) containing 1.0 M NaC1 as
eluent. Bovine serum albumin (66 kDa), carbonic
anhydrase (29 kDa), cytochrome c (12.4 kDa) and
P. furiosus rubredoxin (5.3 kDa; see [3]) were
Thermal stability
T. maritima ferredoxin showed no change in its
U V / v i s i b l e absorption properties or in its electron acceptor activity for pyruvate ferredoxin oxidoreductase (see below) after heating the protein
(1.5 mg m1-1 in 50 mM Tris " HCI, pH 8.0) for 12
167
h at 95°C. After 24 h at this temperature, the
decreased by about 20% indicating some destruction of the FeS chromophore.
The thermal stability of this protein is therefore
similar to that previously reported for P. furiosus
ferredoxin [8]. For comparison, the ferredoxin
from the moderate thermophile Clostridium thermocellum, has a half life (tl/2) at 80°C of about
60 min [11].
A39o/A280 ratio
g = 2.06
i
g = 1,93
I-
g-189
Electron carrier activity
Dithionite-reduced T. maritima ferredoxin did
not function as an electron donor for H e evolution by purified T. rnaritima hydrogenase [5], even
at concentrations up to 0.5 mM and temperatures
up to 90°C, but the oxidized protein did replace
methyl viologen as an electron acceptor for pyruvate oxidation in the routine assay of T. maritirna
pyruvate ferredoxin oxidoreductase [4]. Using
pyruvate (5 raM) and CoA (0.2 mM), the apparent K m and Vmax values were approximately 26
/xM and 280 /~mol of pyruvate oxidized min -~
mg -1, respectively, at 80°C. Similarly, the oxidized ferredoxin was readily reduced at 80°C by
1.5
1.0
,5
<
",,
I
360
P
420
1
I
I
325
350
375
Magnetic Field / m T
Fig. 2. EPR spectrum of reduced T. maritima ferredoxin. The
ferredoxin (6 mg ml - t in 50 mM Tris.HCl, pH 8.0) was
reduced with sodium dithionite (2 mM). The conditions were:
temperature, 6K; microwave power, 10 mW; modulation amplitude, 0.25 mT; microwave frequency, 9.42 GHz.
pyruvate ferredoxin oxidoreductase in the presence of CoA and pyruvate, as its visible absorption spectrum (Fig. 1) was identical to that obtained after reduction of the oxidized protein
with sodium dithionite (data not shown). Fermentative, H2-evolving anaerobic bacteria typically
utilize ferredoxin as the electron carrier between
pyruvate oxidoreductase and an H z-evolving hydrogenase (see [5]). However, T. maritima must
have an additional intermediate electron carrier(s).
Nature of the iron-sulfur cluster
T. maritima ferredoxin reduced by sodium
a
0.5
0.0
I
300
[
480
I
540
600
Wavelength (nm)
Fig. 1. Visible absorption spectra of T. maritirna ferredoxin.
The ferredoxin (0.29 mg m1-1) was in 50 mM N-(2-hydroxyethyl)piperazine N'-3-morpholinopropane sulfonic acid
(EPPS; pH 8.4) containing 0.1 M NaCI. (a) After air oxidation; (b) after incubation with T. maritima pyruvate ferredoxin
oxidoreductase (5 U) in the presence of coenzyme A (0.1
mM), MgCI 2 (1 raM), thiamine pyrophosphate (0.05 mM) and
pyruvate (5 raM) at 80°C for 2 min under anaerobic conditions
(see [4]). Spectra were recorded at 23°C.
dithionite exhibited a rhombic E P R spectrum with
a gz = 2.06, gy = 1.93 and g~ = 1.89 (Fig. 2). This
signal was observed up to 20 K and represented
0.95 _+ 0.14 spins mol-1. No additional absorption
was observed at lower magnetic fields. The spectrum is typical of a magnetically isolated S = 1 / 2
[4Fe-4S] ~+ cluster (see [10]). Together with the
above results, these data show that the ferredoxin
is a monomeric protein containing a single 4Fecluster which in the reduced form exists exclusively with a S = 1 / 2 ground state. Almost all
ferredoxins containing 4Fe-clusters exhibit similar
E P R properties. A notable exception is the pro-
168
Tm:
TI:
Pf:
Ct:
Mt:
Dg:
Bt:
Da:
Av:
Pa:
.MKVR~DADA
.MKVSVDKDA
AWKVSVDQDT
.MKVTVDGDL
.MPALVNADE
..PIE VNDD.
PKYTIVDKET
ARKFYVDQDE
...AFVVTDN
...AYyINDS
CIGC..GVCE
CIGC..GVCA
CIGD..AICA
CIAC..QT~I
CSGC..GSCV
~MAC..EACV
C IAC..~ACG
C IAC..ESCV
C!K~KYTDCV
C__~IAC..~ACK
NLCPDVFQLG
SICPDVFEMD
SLCPDVFEMN
DLCPSVEDWD
DECPSEAITL
EICPDVFEMN
AAAPDIYDYD
EIAPGAFAMD
EV~P.VDCFY
PECP.VNIQ.
D D G K A K .... _____QPETDLPC
D D G K A K .... A L V A E T D L E C
DEGKAQPKVE VIEDEELYN~
pE~SLHVIVD EVPEGAEDS~
DEEKGI.AV. yDQDECVE C
EE~D..KAV. VINPDSDLD~
EDGIAQGIVE V..PDILIDD
............. PEIEKAY
E.HPN...FL VIHPDECID~
Q . H S .... IY A I D A D S C I D ~
AK-(100)
AK- (74)
AK- (58)
AR- (44)
GA- (38)
VE- (35)
MM- (35)
VK- (28)
AL- (28)
GS- (24)
Fig. 3. N-terminal amino acid sequences of T. maritima ferredoxin and representative archaeal and bacterial ferredoxins. Gaps (.)
have been inserted to maximize homology. The abbreviations used and the number of iron atoms each ferredoxin contains are: Tm,
T. maritima (4Fe); Pf, Pyrococcus furiosus (4Fe); TI, Thermococcus litoralis (4Fe); Dg, Desulfovibrio gigas Fd I (3Fe); Bt, Bacillus
thermoproteolyticus (4Fe); Da, D. africanus Fd I (7Fe); Ct, Clostridium thermoaceticum (4Fe); Mt, Methanosarcina thermophila
(8Fe); Av, Azotobacter vinelandii (7Fe); Pa, Peptococcus aerogenes (8Fe). The asterisks denote the cluster-binding residues for the
4Fe-ferredoxins. Numbers in parentheses indicate identity (%) with Tm ferredoxin. Data taken from [9,13,14].
tein from P. furiosus whose reduced 4Fe-center
has a predominantly S = 3 / 2 ground state which
gives rise to an approx, g = 5 EPR signal [12].
This unusual property is thought to be related to
the partial non-cysteinyl coordination to the cluster, a property not shared by the ferredoxin of
the hyperthermophile T. litoralis (see Fig. 3).
N-terminal amino acid sequence analysis
The sequence of the first 44 amino acids of T.
maritima ferredoxin was determined and this is
aligned in Fig. 3 with the corresponding sequences of the two 4Fe-ferredoxins from hyperthermophilic archaea ( P. furiosus and Ts. litoralis;
[9]), together with representatives of the more
than 30 sequences of mesophilic ferredoxins that
are known (see [13]). From the consensus sequence of cysteinyl residues found in these proteins, the single [4Fe-4S] cluster in T. maritima
ferredoxin appears to have complete cysteinyl
coordination, in agreement with the spectroscopic
analysis. Remarkably, the amino acid sequence of
T. maritima ferredoxin (45/61 residues) shows
very high homology with the two ferredoxins (both
of the 4Fe-type) from the hyperthermophilic archaea, e.g. 74% identity with the Thermococcus
protein, but significantly lower homology with
ferredoxins from bacteria and methanogenic archaea (from 19 to 44% comparing all known
sequences; [13,14]). Interestingly, the highest
identities with the non-hyperthermophilic proteins are with the ferredoxins from the moderately thermophilic bacterium, C. thermoaceticum
(44% identity) [11] and from the moderately thermophilic, methanogenic archaeon, Methanosarcina thermophila (38% identity) [14]. However,
systematic analyses of the amino acid sequences
of analogous mesophilic, moderately thermophilic
and hyperthermophilic proteins, even in the case
of small redox proteins, have as yet offered no
clue as to the mechanisms of protein 'hyperthermostability' (see [3]).
The great similarity between the ferredoxins
from hyperthermophilic archaea and the hyperthermophilic bacterium, T. maritima, in amino
acid sequence, thermal stability and cluster content, obviously suggests a close evolutionary relationship between these proteins. The homology is
surprising, however, for two reasons. First, the
ferredoxin-dependent metabolic pathways that
these two groups of organisms use for sugar oxidation are very different (see [3]). Second, these
organisms appear to be the most ancient of all
known life forms [2], yet the ancestral ferredoxin
is thought to be of the 8Fe-type and to contain
two [4Fe-4S] clusters (see [13]). Clearly, the resuits presented here suggest that a revision might
be necessary in the evolutionary mechanisms of
these simple iron-sulfur proteins.
Acknowledgements
This research was supported by grants from
the Department of Energy (FG09-88ER13901)
and by a National Science Foundation Training
169
Group Award to the Center for Metalloenzyme
Studies of the University of Georgia (DIR
9014281).
7
8
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