©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ferredoxin-dependent Redox System of a Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7
PURIFICATION AND CHARACTERIZATION OF A NOVEL REDUCED FERREDOXIN-REOXIDIZING IRON-SULFUR FLAVOPROTEIN (*)

(Received for publication, December 29, 1994; and in revised form, May 22, 1995)

Toshio Iwasaki (§) Takayoshi Wakagi Tairo Oshima (¶)

From the Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 226, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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,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).


INTRODUCTION

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 oxidoreductase(5) . In the case of a hyperthermophilic archaeon Pyrococcus furiosus, the hydrogenase also functions as an elemental sulfur reductase(6) .

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 (100 300 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.

Sulfolobus sp. strain 7 (originally named as Sulfolobus acidocaldarius strain 7)()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) .

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, 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) )).()

In this paper, we report purification and characterization of a novel ferredoxin-linked FeS flavoprotein, IFP,()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.


EXPERIMENTAL PROCEDURES

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.

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.

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.

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) of 0.70(26) . Approximately 30-40 mg of purified material could be routinely obtained from about 150 g (wet weight) of the cells.

A soluble 2-oxoacid:ferredoxin oxidoreductase was also purified from the cytosol fraction to an electrophoretically homogeneous state, as will be described elsewhere. 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.

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 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).

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 cm(31) .

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) .


RESULTS

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 NADPin 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.

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 (, , 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.


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 (::) was estimated to be 0.86:1.0:1.0, respectively, on the basis of the relative intensities.



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 (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).

The sequence of the N-terminal 17 amino acid residues of the 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.


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 = 3.61).





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 = 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).


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/-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) .


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.''



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).


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 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.



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, 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.

Taken together, these data suggest that the relative order of redox potentials of the chromophores in IFP is FMN/FMNH (-57 mV) FMNH/FMNH > [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.


DISCUSSION

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).

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 ) 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.


FOOTNOTES

*
This investigation was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Life Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 226, Japan. Tel.: +81-45-924-5712; Fax: +81-45-924-5805.

Present address: Dept. of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-03, Japan.

The organism had been isolated from Beppu hot springs, Japan, originally named as Sulfolobus acidocaldarius strain 7, but was recently redesignated tentatively as ``Sulfolobus sp. strain 7'' due to small differences in 16 S rRNA base sequences of strain 7 and S. acidocaldarius type strain DSM 639. The preliminary 16 S rRNA sequence analysis suggests that the isolate is a novel species belonging to the genus Sulfolobus.

T. Wakagi, and T. Oshima, manuscript in preparation.

T. Iwasaki, T. Wakagi, and T. Oshima, manuscript in preparation.

Although an extremely slow ferredoxin-dependent NADPH formation was detected at higher pH, the specific activity at pH 7.6 and at 50 °C was only 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).

The abbreviations used are: IFP, reduced ferredoxin-oxidizing iron-sulfur flavoprotein of Sulfolobus sp. strain 7; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-[N-morpholino]-propanesulfonic acid.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Mukund, S., and Adams, M. W. W.(1991)J. Biol. Chem. 266, 14208-14216 [Abstract/Free Full Text]
  2. Adams, M. W. W. (1993)Annu. Rev. Microbiol.47,627-658 [CrossRef][Medline] [Order article via Infotrieve]
  3. Schicho, R. N., Ma, K., Adams, M. W. W., and Kelly, R. M.(1993)J. Bacteriol. 175, 1823-1830 [Abstract]
  4. Bryant, F. O., and Adams, M. W. W.(1989)J. Biol. Chem. 264, 5070-5079 [Abstract/Free Full Text]
  5. Schfer, T., and Schnheit, P.(1993)Arch. Microbiol. 159, 354-363
  6. Ma, K., Schicho, R. H., Kelly, R. M., and Adams, M. W. W.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 5341-5344 [Abstract]
  7. Danson, M. J. (1989)Can. J. Microbiol.35,58-64 [Medline] [Order article via Infotrieve]
  8. Iwasaki, T., Wakagi, T., Oshima, T., and Matsuura, K. (1994) in Flavins and Flavoproteins (Yagi, K., ed) pp. 755-758, Walter de Gruyter, New York
  9. Kerscher, L., Nowitzki, S., and Oesterhelt, D.(1982)Eur. J. Biochem. 128, 223-230 [Abstract]
  10. Kerscher, L., and Oesterhelt, D.(1982)Trends Biochem. Sci. 7, 371-374 [CrossRef]
  11. Koike, M., and Koike, K. (1976)Adv. Biophys.9,187-227
  12. Kerscher, L., and Oesterhelt, D.(1981)Eur. J. Biochem. 116, 587-594 [Abstract]
  13. Blamey, J. M., and Adams, M. W. W.(1993)Biochim. Biophys. Acta 1161, 19-27 [Medline] [Order article via Infotrieve]
  14. Uyeda, K., and Rabinowitz, J. C.(1971)J. Biol. Chem. 246, 3111-3119 [Abstract/Free Full Text]
  15. Gehring, U., and Arnon, D. I.(1972)J. Biol. Chem. 247, 6963-6969 [Abstract/Free Full Text]
  16. Shah, V. K., Stacey, G., and Brill, W. J.(1983)J. Biol. Chem. 258, 12064-12068 [Abstract/Free Full Text]
  17. Wahl, R. C., and Orme-Johnson, W. H.(1987)J. Biol. Chem. 262, 10489-10496 [Abstract/Free Full Text]
  18. Meinecke, B., Bertram, J., and Gottschalk, G.(1989)Arch. Microbiol. 152, 244-250 [Medline] [Order article via Infotrieve]
  19. Blamey, J. M., and Adams, M. W. W.(1994)Biochemistry 33, 1000-1007 [Medline] [Order article via Infotrieve]
  20. Williams, K., Lowe, P. N., and Leadlay, P. F.(1987)Biochem. J. 246, 529-536 [Medline] [Order article via Infotrieve]
  21. Mller, M.(1988)Annu. Rev. Microbiol. 42, 465-488 [CrossRef][Medline] [Order article via Infotrieve]
  22. Kerscher, L., and Oesterhelt, D.(1981)Eur. J. Biochem. 116, 595-600 [Abstract]
  23. Wakagi, T., and Oshima, T.(1986)Syst. Appl. Microbiol. 7, 342-345
  24. Wakao, H., Wakagi, T., and Oshima, T.(1987)J. Biochem. (Tokyo) 102,255-262 [Abstract]
  25. Fujii, T., Moriyama, H., Takenaka, A., Tanaka, N., Wakagi, T., and Oshima, T.(1991) J. Biochem. (Tokyo) 110,472-473 [Abstract]
  26. Iwasaki, T., Wakagi, T., Isogai, Y., Tanaka, K., Iizuka, T., and Oshima, T.(1994) J. Biol. Chem. 269, 29444-29450 [Abstract/Free Full Text]
  27. Moll, R., and Schfer, G.(1988)FEBS Lett. 232, 359-363 [CrossRef]
  28. Mukund, S., and Adams, M. W. W.(1993)J. Biol. Chem. 268, 13592-13600 [Abstract/Free Full Text]
  29. Dutton, P. L. (1978)Methods Enzymol.54,411-435 [Medline] [Order article via Infotrieve]
  30. Faeder, E. J., and Siegel, L. M.(1973)Anal. Biochem. 64, 136-141
  31. Whitby, L. G. (1953)Biochem. J.54,437-442
  32. Laemmli, U. K. (1970)Nature227,680-685 [Medline] [Order article via Infotrieve]
  33. Ortiz, M. L., Calero, M., Patron, C. F., Castellanos, L., and Mendez, E.(1992) FEBS Lett. 296, 300-304 [CrossRef][Medline] [Order article via Infotrieve]
  34. Plaga, W., Lottspeich, F., and Oesterhelt, D.(1992)Eur. J. Biochem. 205, 391-397 [Abstract]
  35. Knaff, D. B., and Hirasawa, M.(1991)Biochim. Biophys. Acta 1056, 93-125 [Medline] [Order article via Infotrieve]
  36. Mason, J. R., and Cammack, R.(1992)Annu. Rev. Microbiol.46,277-305 [CrossRef][Medline] [Order article via Infotrieve]
  37. Malkin, R. (1973) in Iron-Sulfur Proteins (Lovenberg, W., ed) Vol. 2, pp. 1-26, Academic Press, New York
  38. Komai, H., Massey, V., and Palmer, G.(1969)J. Biol. Chem. 244, 1692-1700 [Abstract/Free Full Text]
  39. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., Rawlings, J., Rao, K. K., and Hall, D. O.(1978)Biochemistry 17, 4770-4778 [Medline] [Order article via Infotrieve]
  40. Palmer, G., and Massey, V.(1969)J. Biol. Chem. 244, 2614-2620 [Abstract/Free Full Text]
  41. Porras, A. G., and Palmer, G.(1982)J. Biol. Chem. 257, 11617-11626 [Abstract/Free Full Text]
  42. Hunt, J., Massey, V., Dunham, W. R., and Sands, R. H.(1993)J. Biol. Chem. 268,18685-18691 [Abstract/Free Full Text]
  43. Hille, R. (1992) in Chemistry and Biochemistry of Flavoenzymes (Mueller, F., ed) Vol. III, pp. 21-68, CRC Press, Boca Raton, FL
  44. Tshisuaka, B., Kappl, R., Httermann, J., and Lingens, F.(1993) Biochemistry 32, 12928-12934 [Medline] [Order article via Infotrieve]
  45. Batie, C. J., LaHaie, E., and Ballou, D. P.(1987)J. Biol. Chem. 262, 1510-1518 [Abstract/Free Full Text]
  46. Correll, C. C., Batie, C. J., Ballou, D. P., and Ludwig, M. L.(1992)Science 258,1604-1610 [Medline] [Order article via Infotrieve]
  47. Iwasaki, T., Wakagi, T., and Oshima, T. (1993) International Workshop on Molecular Biology and Biotechnology of Extremophiles and Archaebacteria pp. 69-70 (August 1993, Wako-shi, Japan) (abstr.)

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.