(Received for publication, December 29, 1994; and in revised form, May 22, 1995)
From the
To elucidate the ferredoxin-dependent redox system of the
thermoacidophilic, aerobic archaeon Sulfolobus sp. strain 7, a
novel FeS flavoprotein, which can reoxidize the reduced 7Fe ferredoxin in vitro, has been purified and characterized (designated as
IFP) using the cognate 7Fe ferredoxin and 2-oxoacid:ferredoxin
oxidoreductase, a key enzyme of the archaeal tricarboxylic acid cycle.
IFP consists of three non-identical subunits with apparent molecular
masses of 87, 32, and 22 kDa, respectively, and contains at least two
FMN (E Recent studies on the physiological function of ferredoxins in
several strictly anaerobic archaea (archaebacteria) led to unexpected
findings that oxidized ferredoxin functions as an intermediate electron
acceptor of a variety of key steps in the central metabolic pathways
involved in saccharolytic and peptide fermentation, where NAD(P)
usually substitutes in some bacteria
(eubacteria)(1, 2, 3) . Reduced ferredoxin
thus formed is then reoxidized by a hydrogenase (4) and
possibly by a ferredoxin:NADP On the other hand, the
ferredoxin-dependent redox system of aerobic respiratory archaea such
as Halobacteria and Sulfolobus remains only poorly
understood. As in the cases of aerobic bacteria and eukarya
(eukaryotes) with mitochondria, these archaea acquire biological energy
by aerobic respiration rather than simple fermentative pathway, and in
fact contain a membrane-bound aerobic respiratory chain, which directly
couple to an archaeal tricarboxylic acid cycle at least at the level of
the respiratory complex II (succinate:quinone oxidoreductase
complex(7, 8) ). Their oxidative tricarboxylic acid
cycle, however, differs considerably from those of eukarya with
mitochondria and most of aerobic bacteria in that the decarboxylation
of 2-oxoglutarate is catalyzed by a coenzyme-A-acylating
2-oxoacid:ferredoxin oxidoreductase(7, 9, 10) instead of conventional 2-oxoacid dehydrogenase
multienzyme complexes (11) . The archaeal 2-oxoacid:ferredoxin
oxidoreductases are considerably smaller enzymes (100 Sulfolobus sp. strain 7
(originally named as Sulfolobus acidocaldarius strain 7) To elucidate what reoxidizes reduced ferredoxin in Sulfolobus sp. strain 7, we have also partially purified the archaeal
NADPH-specific diaphorase with an NADPH:ferredoxin oxidoreductase
activity; however, as opposed to the case proposed for a
hyperthermophilic archaeon P. furiosus(5) , the in
vitro reconstitution system containing the Sulfolobus 2-oxoacid:ferredoxin oxidoreductase, 7Fe ferredoxin, and
NADPH:ferredoxin oxidoreductase in the presence of
NADP In this
paper, we report purification and characterization of a novel
ferredoxin-linked FeS flavoprotein, IFP,
Aldehyde oxidoreductase activities were tested
spectrophotometrically as described by Mukund and
Adams(1, 28) , except in 10 mM potassium
phosphate buffer, pH 6.0-7.5, at 50 °C.
The Sulfolobus 7Fe ferredoxin
was purified as described previously (25, 26) and was
stored at -80 °C until use. Purified ferredoxin gave a single
band on 20% analytical gel electrophoresis and had a purity index (A A soluble 2-oxoacid:ferredoxin oxidoreductase was also purified from
the cytosol fraction to an electrophoretically homogeneous state, as
will be described elsewhere.
The potentiometric titration of the redox centers of
the purified red FeS flavoprotein (IFP) was performed at room
temperature in a Thunberg-type cell similar to that described by Dutton (29) under continuous flow of N The apparent
molecular masses of the purified proteins were estimated by a gel
filtration analysis with a calibrated Tosoh G-3000XL column equipped
with a Tosoh HPLC 8030, which had been equilibrated with 50 mM MOPS-NaOH buffer, pH 6.8, containing 200 mM NaCl. Protein
was measured by the BCA assay (Pierce Chemical) with bovine serum
albumin as a standard. The metal content analyses of the purified
proteins were carried out by inductively coupled plasma atomic emission
spectrometry with a Seiko SPS 1500 VR instrument. The type of flavin
released from purified IFP by denaturation in the presence of 5%
trichloroacetic acid was identified according to the method of Faeder
and Siegel (30) using a Shimadzu spectrofluorophotometer
RF-540, and the content of FMN was estimated using an extinction
coefficient at 450 nm of 12.2 mM Polyacrylamide gel
electrophoresis in the presence of SDS (SDS-PAGE) was carried out
according to Laemmli (32) on 12-20% gels after treating
proteins with 2% SDS in the presence or absence of 2% 2-mercaptoethanol
at 90 °C for 15 min, and proteins were visualized by Coomassie
Brilliant Blue staining and scanned by a densitometer (Shimadzu
dual-wavelength scanner CS-930). Analytical PAGE (in the absence of
SDS) was carried out with the Laemmli discontinuous system on 5 or 7.5%
gels in the absence of SDS, and proteins were visualized by CBB
staining. N-terminal amino acid sequence analysis of each subunit of
purified IFP was carried out by a 470A Protein Sequencer (Applied
Biosystems), after separation of subunits on SDS-PAGE gels and
recovering by electroelution in the presence of 0.1% SDS; the gels had
been visualized by reversible imidazole/Zn/SDS-staining(33) .
Since various
ferredoxin-dependent redox systems in bacteria and anaerobic archaea
involve numerous FeS proteins(2, 35, 36) ,
several FeS proteins were systematically searched after fractionation
of the Sulfolobus cytosol fraction with a DEAE-Sephacel column
and tested for their abilities to reoxidize enzymatically reduced
ferredoxin; among them, a red FeS flavoprotein copurified with the
cognate 2-oxoacid:ferredoxin oxidoreductase showed a weak
ferredoxin-reoxidizing activity, which was subsequently purified to an
electrophoretically homogeneous state and was tentatively designated as
IFP (see below). Purified IFP showed a single band on 5% analytical
PAGE in the absence of SDS and had an apparent molecular mass of 290
kDa by the gel filtration analysis (data not shown). It consisted of
three dissimilar subunits (
Figure 1:
Polyacrylamide gel electrophoresis in
the presence of SDS of purified IFP from Sulfolobus sp. strain
7 analyzed by densitometric scan. Purified enzyme was analyzed on 13%
SDS-PAGE, stained with Coomassie Brillient Blue, and scanned by a
densitometer. An apparent stoichiometry of the subunits
(
The presence of non-heme iron in purified
IFP was indicated by its red color and was confirmed by the inductively
coupled plasma atomic emission spectrometry, which resulted 14.5 nmol
of Fe/mg of protein ( The sequence of
the N-terminal 17 amino acid residues of the
Figure 2:
UV visible spectra of purified IFP of Sulfolobus sp. strain 7. The protein concentration was 3.7
mg/ml in 20 mM potassium phosphate buffer, pH 6.8. The scale
of the full range spectrum of the oxidized IFP is reduced to 0.2-fold (viz., A
Fig. 3shows the CD spectra of the air-oxidized and
dithionite-reduced IFP. The oxidized protein has two negative troughs
at 378 and 548 nm, a dominant positive peak at 430 nm, another positive
feature around 470 nm, and at least two weak positive peaks between 550
and 800 nm (Fig. 3, trace A). The reduced protein has
two weak positive peaks at 402 and 524 nm, at least one weak broad
positive peak between 580 and 800 nm, and a negative trough at 457 nm (Fig. 3, trace B). The intense positive peak in the
region around 430 nm, where a flavin contributes only slightly (38) , is strictly characteristic of the oxidized
plant-ferredoxin-type [2Fe-2S] cluster (39, 40) (see below).
Figure 3:
Circular dichroism spectra of purified IFP
of Sulfolobus sp. strain 7. The protein concentration was as
in Fig. 2.
Fig. 4shows the
X-band EPR spectrum at 37 K of dithionite-reduced IFP. It elicits a
rhombic EPR signal at g
Figure 4:
EPR spectrum of dithionite-reduced IFP of Sulfolobus sp. strain 7. The protein concentration was as in Fig. 2. Instrument settings: temperature, 37 K; microwave power,
2 milliwatts; modulation amplitude, 0.79 milliTesla; the g values are indicated in the figure.
Thus, these data suggest the
presence of at least two each of non-covalently bound FMN and
plant-ferredoxin-type [2Fe-2S]
centers/
Figure 5:
Potentiometric titration of the Sulfolobus IFP (left) and difference spectra measured
in three different ranges of ambient redox potentials (right).
The ambient redox potential was changed by adding a small amount of
ditionite solution. For conditions, see ``Experimental
Procedures.''
Figure 6:
Ferredoxin-dependent anaerobic reduction
of the Sulfolobus IFP. The reaction mixture (containing 2
mM 2-oxoglutarate, 100 µM coenzyme A, and 8.6
µg of purified 2-oxoacid:ferredoxin oxidoreductase
Careful spectral analysis of the time course of the
ferredoxin-dependent reduction of IFP (Fig. 6A) at 420,
460, and 550 nm suggests that the reduction of the prosthetic groups
proceeds at least in three different steps (Fig. 6B) (cf. Refs.(41, 42) ). All of the initial
absorbance at 550 nm is due to the [2Fe-2S] center, while
those at 460 and 420 nm are due to both [2Fe-2S] and FMN
centers. During the initial 60 min after addition of the cognate
ferredoxin, Taken
together, these data suggest that the relative order of redox
potentials of the chromophores in IFP is FMN/FMNH (-57 mV)
FMNH/FMNH
It is currently not possible to assign a true physiological function
of IFP merely by analogy to the other ferredoxin-dependent systems of
anaerobic archaea and bacteria. While the subunit structure and/or the
cofactor composition of IFP indicate possible similarities to those of
nonheme-iron-containing hydroxylases, such as aldehyde oxidoreductases,
xanthine oxidase/dehydrogenases, and a bacterial quinoline
2-oxidoreductase(43, 44) , neither IFP nor the cytosol
fraction of Sulfolobus sp. strain 7 showed any
ferredoxin-dependent formaldehyde/acetaldehyde/benzoaldehyde
oxidoreductase activity at pH 6.8 and at 50 °C (data not shown).
The cofactor composition of purified IFP is also similar to those of
NADH-dependent dioxygenase reductase components of several bacterial
dioxygenase systems(36, 45, 46) , but their
intramolecular electron transfer scheme ((NADH
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,6.8 = -57 mV) and two
plant-ferredoxin-type [2Fe-2S]
clusters (E
,6.8 = -260
mV)/
structure. Both
FeS and flavin centers of IFP are slowly but fully reduced by the
enzymatically reduced cognate ferredoxin under anaerobic conditions at
50 °C, but not by NAD(P)H. Thus, the ferredoxin-dependent redox
system of Sulfolobus sp. strain 7 is tentatively proposed as
follows: 2-oxoacid:ferredoxin oxidoreductase (thiamine pyrophosphate
and [4Fe-4S] cluster)
ferredoxin
IFP
([2Fe-2S] cluster
FMN).
oxidoreductase(5) . In the case of a hyperthermophilic
archaeon Pyrococcus furiosus, the hydrogenase also functions
as an elemental sulfur reductase(6) .
300 kDa)
and contain at least thiamine pyrophosphate and
[4Fe-4S]
cluster as prosthetic
groups(10, 12, 13) . These enzymes catalyze
in the 2-oxoacid-dependent reduction of ferredoxins, but do not utilize
NAD(P)
as a physiological electron acceptor.
Interestingly, their cofactor composition is remarkably similar to
those from fermentative and phototrophic obligatory anaerobic bacteria (14, 15, 16, 17, 18, 19) and
anaerobic and amitochondrial protozoa(20, 21) .
However, as opposed to the cases proposed in earlier studies for some
obligatory anaerobes such as Clostridia and Chlorobia(7, 14, 15) , Halobacterium salinarium enzymes probably do not catalyze
``reverse'' 2-oxoacid synthase reaction for reoxidation of
reduced ferredoxin and CO
fixation(12, 22) . In fact, the reoxidation
process of enzymatically reduced ferredoxin in aerobic respiratory
archaea remains unknown.
(
)is a typical aerobic and thermoacidophilic archaeon
isolated from Beppu hot springs, Kyushu, Japan, and grows optimally at
pH 2-3 and at 75-80 °C. The chemoheterotrophically
grown archaeon acquires biological energy by aerobic respiration rather
than simple fermentative pathway(8, 23) , and contains
several membrane-bound respiratory proteins such as an NADH
dehydrogenase(24) , a succinate:quinone oxidoreductase complex,
and an unusual respiratory terminal oxidase complex(8) . On the
other hand, the archaeal cytoplasmic fraction contains a large amount
of a 7Fe ferredoxin, which has been purified,
crystallized(25) , and sequenced.
(
)The
subsequent characterization showed that it contains one
[3Fe-4S]
cluster (E =
-280 mV versus NHE) and one
[4Fe-4S]
cluster (E = -530 mV), of which only the [3Fe-4S]
cluster is reduced during the steady-state turnover of the cognate
2-oxoacid:ferredoxin oxidoreductase(26) . The presence of this
enzyme in Sulfolobus had been predicted by Kerscher et
al. (9) and was purified from Sulfolobus sp.
strain 7 and characterized recently. It appears to be the most similar
to H. salinarium pyruvate:ferredoxin oxidoreductase (12, 34) in terms of cofactor composition (one
thiamine pyrophosphate and one low potential
[4Fe-4S]
cluster) and broad
substrate specificities toward 2-oxoacids (for the Sulfolobus enzyme, the K
for 2-oxoglutarate
= 0.87 mM; the K
for
pyruvate = 0.25 mM).
(
)The
enzymes from other sources are specific to either pyruvate or
2-oxoglutarate (12, 13, 17, 18, 19) .
, 2-oxoacid, and CoA did not catalyze any
2-oxoacid- and ferredoxin-dependent formation of NADPH when tested
anaerobically at 50 °C and at physiological pH (pH 5.5-6.8 (cf. Ref.(27) )).
(
)
(
)from Sulfolobus sp. strain 7. IFP contains two
[2Fe-2S]
clusters and two FMN as
prosthetic groups in an
structure and is most likely involved in the reoxidation process
of the Sulfolobus ferredoxin which has been reduced
enzymatically by the cognate 2-oxoacid:ferredoxin oxidoreductase.
Materials
Sodium pyruvate and 2-oxoglutarate
were from Nakarai Tesque (Tokyo). CoA was obtained from Kohjin (Tokyo).
DEAE-Sephacel, phenyl-Toyopearl 650 M, and hydroxylapatite HTP
were purchased from Pharmacia LKB Biotechnology Inc., Tosoh
Corporation, and Bio-Rad, respectively. Water was purified by the
Milli-Q purification system (Millipore). Other chemicals mentioned in
this study were of analytical grade.Measurement of Enzymatic
Activities
2-Oxoacid:ferredoxin oxidoreductase activities were
monitored with the horse heart cytochrome c reduction assay at
50 °C using the purified Sulfolobus dicluster ferredoxin
as an intermediate electron acceptor(9) , except that the
reaction was carried out in 10 mM potassium phosphate buffer,
pH 6.8, and was initiated by addition of the purified enzyme.
Non-enzymatic reduction of cytochrome c by coenzyme A at this
temperature was corrected. For routine assays, 2-oxoacids
(2-oxoglutarate was mainly used), coenzyme A, the Sulfolobus ferredoxin, and horse heart cytochrome c (Sigma) were
present at the concentrations of 2-4 mM, 50-100
µM, 25 µM, and 50 µM,
respectively.Purification of a 7Fe Ferredoxin and a
2-Oxoacid:Ferredoxin Oxidoreductase from Sulfolobus sp. Strain
7
Sulfolobus sp. strain 7 was cultivated
aerobically and chemoheterotrophically at pH 2.5-3 and
75-80 °C as described previously (23, 24) and was harvested in the late exponential
phase of growth and stored at -80 °C until use. The cells
were suspended in 40 mM Tris-Cl buffer, pH 7.5, containing 0.5
mM phenylmethylsulfonyl fluoride and 1 mM EDTA and
disrupted with a French press (Otake Works, Tokyo) at 1500 kg/cm
twice, and the supernatant (containing negligible amount of
cytochrome) obtained after ultracentrifugation with a Beckman 45Ti
rotor at 130,000
g for 100 min at 15 °C, was used
as the cytosolic fraction.
/A
) of
0.70(26) . Approximately 30-40 mg of purified material
could be routinely obtained from about 150 g (wet weight) of the cells.
Approximately 6-8 mg of
pure enzyme could be obtained from about 100 g (wet weight) of the
cells, and the purified enzyme was stored at -80 °C until
use. The purified enzyme used in this study had the specific activities
of 69 units/mg with 2-oxoglutarate and 39 units/mg with pyruvate,
respectively.
Purification of a Novel Red Iron-Sulfur Flavoprotein
(IFP) from Sulfolobus Strain 7
All steps were carried out at
room temperature unless otherwise specified and by following three
different absorption bands at 280, 430, and 570 nm and the patterns on
SDS-PAGE. The cytosol fraction was applied to a DEAE-Sephacel column
(3.0 28 cm) which had been previously equilibrated with 30
mM Tris-Cl buffer, pH 7.3, containing 1 mM EDTA and 1
mM phenylmethylsulfonyl fluoride. The column was then washed
with 500 ml of the equilibration buffer, and the materials bound to the
column was eluted by a 1600-ml linear gradient of NaCl (0-400
mM) in the equilibration buffer. The red fractions eluted
around 50 mM NaCl were collected and combined, and then made
to 1 M ammonium sulfate solution by addition of solid ammonium
sulfate at 4 °C, while gently stirring. The suspension was gently
degassed by aspirator at room temperature and was directly applied to a
phenyl-Toyopearl 650 M column (1.2
25 cm) that had
been pre-equilibrated with 20 mM potassium phosphate buffer,
pH 6.8, containing 1 M ammonium sulfate. The column was washed
with 100 ml of the equilibration buffer, and a 200-ml linear gradient
was run between 1 M and 0 M ammonium sulfate in 20
mM potassium phosphate buffer, pH 6.8. At this step, the
red-colored fractions eluted at the end of the linear gradient were
clearly separated from the 2-oxoacid:ferredoxin oxidoreductase and
diaphorase (NAD(P)H:dye oxidoreductase) activities. When necessary, the
column was further washed by 20 mM potassium phosphate buffer,
pH 6.8, to enhance the complete elution of the red protein. The
combined fractions were then diluted 5-fold by addition of 20 mM potassium phosphate buffer, pH 6.8, and applied to a Bio-Rad
hydroxylapatite column (1.2
23 cm) which had been
pre-equilibrated with distilled water (Milli-Q). The column was washed
with 100 ml of 10 mM potassium phosphate buffer, pH 6.8, and
the red protein still bound to the column was eluted by a 250-ml linear
gradient of potassium phosphate buffer, pH 6.8 (10-200
mM). The red protein eluted around 110 mM potassium
phosphate was collected, and was concentrated by pressure filtration on
an Amicon YM-10 membrane under N
gas at 4 °C. The
concentrated material (3 ml or less) was loaded onto a 34-ml glycerol
density gradient (10-24%, v/v) in 20 mM potassium
phosphate buffer, pH 6.8, which had been placed on 0.5 ml of 25%
glycerol (v/v) cushion (containing 20 mM potassium phosphate
buffer, pH 6.8), and was ultracentrifugated with a Beckman SW28 rotor
at 28,000 revolutions/min for 20 h at 10 °C. The red FeS
flavoprotein appeared as a single band at
19% glycerol (v/v),
which was collected by tubing and analyzed by SDS-PAGE for purity. The
purified material thus obtained was tentatively designated as IFP
hereafter, and was stored at -80 °C until use.
Analytical Methods
Absorption spectra were
recorded with a Hitachi U-3210 spectrophotometer equipped with a
thermoelectric cell holder, or with a Beckman DU7400 photodiode-array
spectrophotometer. EPR measurements were carried out using a JEOL
JEX-RE1X spectrometer equipped with an Air Products model LTR-3
Heli-Tran cryostat system, in which temperature was monitored with a
Scientific Instruments series 5500 temperature indicator/controller. CD
spectra were recorded at room temperature on a JASCO J-500C
spectropolarimeter equipped with a JASCO Data Processor DP-50, in
0.5-cm cells. gas and stirring,
in the presence of 5 µM each of methyl viologen,
anthraquinone-
-sulfonic acid, phenadine methosulfate, and vitamin K
as redox mediators. Ambient redox potentials (E
) were monitored with a Pt-Ag/AgCl
electrode (Type PS-165F, Toa Electronic Ltd., Tokyo), and desired
potentials were attained by adding a small volume of potassium
ferricyanide or sodium dithionite solution. The obtained absorption
spectra were recorded with a Shimadzu UV-3000 spectrophotometer
connected to a Fujitsu FM 16
HD-II personal computer. To minimize
the effects of mediators, absorbance pair at 420 and 490 nm were used
for corrections of the base-line drift; the absorbance at 440 and 465
nm of each difference spectrum (after a subtraction of each base line)
were then analyzed for the calculation of the midpoint redox potentials
from the titration curves obtained by a fitting program (written at the
Department of Biology, Tokyo Metropolitan University).
cm
(31) .
Purification and Molecular Composition of a Novel
Iron-Sulfur Flavoprotein of Sulfolobus, IFP
In an earlier stage
of the present studies, a 2-oxoacid:ferredoxin oxidoreductase with
broad substrate specificities toward 2-oxoacids (the specific
activities of 69 units/mg with 2-oxoglutarate (K = 0.87 mM) and 39 units/mg with pyruvate (K
= 0.25 mM),
respectively) was purified from Sulfolobus sp. strain 7,
and appeared to reduce only the [3Fe-4S] cluster of the
cognate 7Fe ferredoxin during the steady-state turnover of the
enzyme(26) . It is the most similar to H. salinarium pyruvate:ferredoxin oxidoreductase (12, 34) and
contains one thiamine pyrophosphate cofactor and one low potential
[4Fe-4S]
cluster.
In
subsequent studies, the archaeal NADPH:ferredoxin oxidoreductase was
partially purified to test for its ability to reoxidize the reduced
form of the cognate 7Fe ferredoxin in the presence of NADP
in vitro. However, the Sulfolobus NADPH:ferredoxin oxidoreductase could not reoxidize ferredoxin
that had been reduced enzymatically by 2-oxoacid:ferredoxin
oxidoreductase in the presence of 2-oxoglutarate and CoA; no NADPH
production could be detected at physiological pH (data not
shown).
It therefore seemed plausible that certain other
enzyme(s) should reoxidize the enzymatically reduced 7Fe ferredoxin in vivo to excerpt the excess reductant formed during the
2-oxoacid oxidation process of Sulfolobus.
,
, and
) on SDS-PAGE with
apparent molecular masses of 87, 32, and 19 kDa, respectively(roughly
with a 1:1:1 stoichiometry; Fig. 1). These data suggest that IFP
is most likely an
-hexamer.
Approximately 50-60 mg of pure protein was reproducibly obtained
from
2000 mg of the cytosolic proteins (corresponding to
60 g
(wet weight) of the cells), indicating that it comprises at least
3% of the total cytosolic proteins of Sulfolobus sp.
strain 7. The apparent abundance may imply a physiological importance
of IFP in the archaeon.
:
:
) was estimated to be 0.86:1.0:1.0, respectively, on
the basis of the relative intensities.
4.2 mol of Fe/mol of protein). The
inductively coupled plasma atomic emission spectrometry also showed
that as isolated IFP did not contain any of the following metals:
cobalt, nickel, tungsten, and zinc (data not shown).
subunit of IFP was
determined to be MLV(R)PGEKVKI(S)VXVNG,
where X and letters in parentheses represent ambiguous amino
acid residues. The partial amino acid sequence search against the EMBL
and Swiss-Prot amino acid sequence data bases, however, did not show
any specific motif or considerable homology to any known sequence for
other metalloenzymes (data not shown), and did not give any clue to
assess a possible function of IFP. On the other hand, the N-terminal
amino acid sequences of the
and
subunits were most likely
blocked, as indicated by extremely low recoveries from the protein
sequencer (data not shown).
Spectral Properties and Potentiometric Titration of the
Sulfolobus IFP
Fig. 2shows the optical spectra of
purified IFP. The air-oxidized spectrum (as prepared) shows absorption
maxima at 278, 341, and 428 nm (the A/A
ratio to be 0.22) and
shoulders at 462 and 547 nm (Fig. 2, trace A). Upon
reduction by excess dithionite, at least two shoulders at 455 and 540
nm are observed (Fig. 2, trace C). The optical
properties resemble those of FeS proteins containing a
plant-ferredoxin-type [2Fe-2S] cluster(37) . A
distinct shoulder at 462 nm in the air-oxidized state (trace
B) indicates the presence of flavin, which has been determined to
be 1.5-1.6 mol of FMN/mol protein after denaturation of purified
IFP in the presence of 5% trichloroacetic acid for 15 min (Table 1). FMN was not efficiently released from the protein by
conventional heat treatment due likely to its extreme thermostability,
which is consistent with the optimal temperature for the growth of Sulfolobus sp. strain 7 (75-80 °C(23) );
only
30% of the absorbance of IFP was bleached without any
substantial change over the visible region, even after boiling the
sample at 100 °C for 10 min (data not shown). On the other hand,
the material precipitated by centrifugation after the acid denaturation
was white, indicating the absence of covalently bound flavin in IFP.
=
3.61).
= 2.01, 1.93,
1.90 (the average g factor, g
=
1.95), which could be detected at least up to 70 K. Quantitation of
this EPR signal by double integration indicated
1 spin/2 Fe, in
conjunction with the iron content analysis (4.2 mol of Fe/mol of
protein). Therefore, the g
= 1.95 signal
can be attributed to a reduced plant ferredoxin-type [2Fe-2S]
cluster, being in line with the CD spectra (Fig. 3). The
air-oxidized form of IFP (as isolated) elicits a weak radical feature
at g =
2.0. No other resonance could be detected
in the low field region, indicating the absence of any rubredoxin-type
ferric atom center (data not shown).
-hexamer in
IFP. Further characterization of the redox centers of IFP were made
spectrophotometrically by the room temperature potentiometric titration
in a Thunberg-type cell(29) , to avoid the complications caused
at cryogenic temperatures as discussed by Porras and Palmer (41) . For calculations of the midpoint redox potentials at pH
6.8 of the FMN/FMNH and the [2Fe-2S]
couples, the absorption pair at 490 and 420 nm was used for the
base-line correction, and the absorbance at 465 nm was analyzed for the
flavin and the FeS centers, which has been successfully fit to
Nernstian, single-electron ``n = 1'' curves (Fig. 5). For reasons discussed below, two n = 1
components at -57 and -260 mV were assigned to the midpoint
redox potentials at pH 6.8 of the FMN/FMNH and the
[2Fe-2S]
couples, respectively. It
was not possible to calculate a midpoint redox potential for the
FMNH/FMNH
couple by this method because of significant
contributions of mediator dyes to absorbance at 600-700 nm
region, and of no contribution of FMNH
to the absorbance
around 460 nm(41, 42) .
Ferredoxin-dependent Reduction of the Prosthetic Groups
of IFP at 50 °C
Fig. 6shows an ability of purified
IFP to reoxidize the enzymatically reduced form of the cognate
ferredoxin under the anaerobic conditions at pH 6.8 and at 50 °C.
Because of the presence of 2 mM 2-oxoacid, 100 µM CoA, and only catalytic amounts of purified 2-oxoacid:ferredoxin
oxidoreductase (8.6 µg) and ferredoxin (27 µg), both 2-oxoacid:ferredoxin oxidoreductase and ferredoxin undergo the
steady-state turnover only when an appropriate terminal
electron acceptor is present in the in vitro system. Under the
steady-state conditions, the direct reduction behavior of the
prosthetic groups of the terminal acceptor can be readily monitored
spectrophotometrically. As shown in Fig. 6, A and B, the complete reduction of both prosthetic groups of IFP
(1.23 mg/ml) was achieved in the ferredoxin-dependent manner at pH 6.8
and at 50 °C. The rate of reduction of IFP became negligible in the
absence of the Sulfolobus ferredoxin (Fig. 6B), or when either NADH or NADPH (each 80
µM) was used instead of ferredoxin (data not shown).
and
3.8 mg of purified IFP in 30 mM potassium phosphate buffer, pH
6.8, in a final volume of 3.1 ml) was kept anaerobically in the
presence of N
gas and at 50 °C, and the reaction was
initiated by adding a small amount (27 µg) of the purified Sulfolobus ferredoxin. The apparent ratio of
[2-oxoacid/ferredoxin
oxidoreductase]/[ferredoxin]/[IFP] was
1:28:160 (mol/mol/mol), respectively, in this system. A,
traces of the absorption spectrum of the oxidized IFP (trace
2), and its time-dependent spectral changes scanned every 10 min
after addition of purified ferredoxin (traces 3-24; the
spectral features of trace 24 is the same as the fully reduced spectrum
shown in Fig. 2). Scan rate, 300 nm/min; t, time. B, time course of the absorbance changes of the
ferredoxin-reduced minus fully reduced difference spectra of
IFP measured at three different wavelength (420, 460, and 550 nm, bottom panel); the conditions are the same as in A:
the top panel shows a control experiment measured at 460 nm in
the absence of ferredoxin. y axis, a decrement of absorbance (
A;
A
of 0.09 corresponds
100% reduction of IFP). The top panel shows a control
experiment measured at 460 nm in the absence of ferredoxin. C,
selected ferredoxin-reduced minus fully reduced difference
spectra of A.
40% of the total reducible absorbance at 460 nm is
lost, while the decrement of absorbance at 550 nm was trace due likely
to a formation of the long-wavelength charge-transfer complex (cf. Refs.(41, 42) ). The resulting
difference spectrum (Fig. 6C, dot-dash) has
absorption peaks at 420 and 465 nm, clearly indicating that a bulk of
the [2Fe-2S] cluster of IFP remains in the oxidized state
under the conditions. Thus, while the [2Fe-2S] center is
gradually reduced, causing a decrease in absorbance both at 460 and 550
nm, and a flavin semiquinone (FMNH) is probably formed at this stage,
causing the corresponding increase at 550 nm. During the following 80
min (60-140 min, Fig. 6B), absorbance at 550 nm
decreased, indicating a loss of the semiquinone (and the resulting
formation of FMNH
) and further reduction of the
[2Fe-2S] center, as more electron is transferred from
2-oxoacid via ferredoxin. Consequently,
83% of the total reducible
absorbance at 460 nm is lost (Fig. 6B), and the
difference spectrum suggests the presence of only a small amount of the
oxidized [2Fe-2S] center (Fig. 6C, dotted
line). Semiquinone form of FMN is apparently absent in the
spectrum. Therefore, the final step in Fig. 6B (140-240 min) is due likely to slow reduction of the remaining oxidized [2Fe-2S] cluster of IFP.
> [2Fe-2S]
(-260 mV) under the experimental conditions (50 °C, pH
6.8). Hence, the electron transfer from a
[3Fe-4S]
cluster (-280 mV) of the Sulfolobus 7Fe ferredoxin (26) probably occurs as
follows: ferredoxin
IFP ([2Fe-2S]
FMN).
Although the midpoint redox potential of the
[3Fe-4S]
center of the Sulfolobus 7Fe ferredoxin (-280 mV(26) ) is close to that of
the NAD
/NADH couple (-320 mV), purified IFP was
partially reduced by the reduced Sulfolobus ferredoxin, but not by 80 µM NAD(P)H at room temperature (data
not shown). These data suggest that a reduction of IFP by the cognate
ferredoxin is probably specific and may not be artificial electron
transfer in vitro.
Molecular Properties of IFP, a Novel Iron-sulfur
Flavoprotein of Sulfolobus
As far as we are aware, there is no
report describing the fate of reduced ferredoxin in aerobic respiratory
archaea. The present paper describes purification and characterization
of a novel FeS flavoprotein, IFP, involved in the ferredoxin-dependent
redox system of the thermoacidophilic respiratory archaeon, Sulfolobus sp. strain 7. The spectral data suggest that IFP
contains two each of nominally identical non-covalently bound FMN
(-57 mV) cofactors and plant-ferredoxin-type [2Fe-2S]
clusters (-260 mV) in
structure, and that
it is probably involved in the reoxidation process of reduced
ferredoxin in Sulfolobus sp. strain 7 as judged by the
following evidence: (i) both FMN and [2Fe-2S] centers of IFP
can be fully reduced in the ferredoxin-dependent manner during
steady-state turnover of 2-oxoacid:ferredoxin oxidoreductase which
keeps the Sulfolobus ferredoxin in the reduced state, when
tested anaerobically at 50 °C. (ii) The potentiometric titration of
IFP suggests that the midpoint redox potential of the
[2Fe-2S] cluster (-260 mV) and the flavin
half-potential (-57 mV) are higher than that of the
redox active [3Fe-4S] cluster of the Sulfolobus ferredoxin (-280 mV)(26) , which is
thermodynamically favorable. (iii) Although NAD(P)H (
-320
mV) was predicted to be able to reduce IFP in terms of thermodynamic
consideration, reduction of IFP in fact occurred in a
ferredoxin-specific manner in vitro. Although a rate of
reduction of a [2Fe-2S] cluster of IFP by the cognate 7Fe
ferredoxin was slow in the in vitro assay system (Fig. 6), it is consistent with the fact that the midpoint
potential of a [2Fe-2S]
cluster of
IFP (-260 mV) is close to that of the redox active
[3Fe-4S]
cluster of the Sulfolobus ferredoxin (-280 mV(26) ). In addition, it may be
accelerated at higher temperatures (50 °C for the present
experiments versus 75-80 °C for the optimal growth
of Sulfolobus(23) ), and an increment of the
[ferredoxin]/[IFP] ratio in vivo (at least
10 times higher than that used for the present experiments on the basis
of a rough estimate of the amounts of the purified proteins; data not
shown). We therefore propose a tentative electron transfer chain of the
cytoplasmic ferredoxin-dependent redox system of Sulfolobus sp. strain 7 as follows: 2-oxoacid:ferredoxin oxidoreductase
(thiamine pyrophosphate and [4Fe-4S] cluster)
ferredoxin
IFP ([2Fe-2S] cluster and FMN)
a putative terminal electron acceptor or metabolic intermediate
(with a redox couple probably well above -160 mV at neutral pH).
) FMN
[2Fe-2S]) differs considerably from that of IFP proposed in
this study ((reduced ferredoxin
) [2Fe-2S]
FMN).
Implication for the Central Metabolism of
Sulfolobus
In earlier reports, it has been reported that
2-oxoacid:ferredoxin oxidoreductases from obligatory anaerobic and
fermentative bacteria could function as a ``reverse''
2-oxoacid synthase to serve for CO fixation(14) .
By analogy, it has been proposed that certain autotrophically grown
aerobic respiratory archaea possess ``reductive''
tricarboxylic acid pathway, which assimilates CO
at the
level of 2-oxoglutarate(7) , although this has not been
conclusively demonstrated experimentally and is thermodynamically
unfavorable at least with the ferredoxin-dependent redox system of H. salinarium(22) . We have recently shown that the Sulfolobus dicluster-type ferredoxin contains one
[3Fe-4S]
cluster (-280 mV) and one
[4Fe-4S]
cluster (-530 mV)
and that only the former redox center is involved in the redox function
during the steady-state turnover of the cognate 2-oxoacid:ferredoxin
oxidoreductase(26) , which has a dual function as the
CoA-dependent pyruvate oxidation and 2-oxoglutarate oxidation as a
member of the archaeal tricarboxylic acid cycle in
chemoheterotrophically grown Sulfolobus sp. strain 7.
Since the archaeal tricarboxylic acid cycle directly couples to
the active membrane-bound aerobic respiratory chain at the level of
succinate(8, 23) , the tentative ferredoxin-dependent
electron transport system proposed in this paper predicts that two
electrons donated from a 2-oxoacid oxidation reaction are efficiently
transferred to ferredoxin and further downstream to IFP; the abundance
of IFP in the cytoplasm of Sulfolobus sp. strain 7 is in line
with this proposal. Further studies are underway to search for the
physiological substrate(s) of IFP and to elucidate the overview of the
archaeal ferredoxin-dependent redox system.
2% of an NADPH:ferredoxin
oxidoreductase activity at the same pH and temperature, indicating that
it is unlikely of physiological importance ((47) ; T. Iwasaki,
and T. Oshima, unpublished data).
We thank Drs. Y. Isogai and T. Iizuka (The RIKEN
Institute) for measuring the EPR spectra, Dr. K. Matsuura (Tokyo
Metropolitan University) for help in the redox titration of IFP, Drs.
T. Fujiwara and Y. Fukumori (Tokyo Institute of Technology) for running
the automatic gas-phase protein sequencer, and Dr. N. Wakiya (Tokyo
Institute of Technology) for metal content analysis by the ICP atomic
emission spectrometry. We also thank Drs. M.-H. Sato and N. Hirohashi
(Tokyo Institute of Technology) for their valuable technical advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.