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J. Biol. Chem., Vol. 275, Issue 33, 25391-25401, August 18, 2000

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Spectroscopic Investigation of Selective Cluster Conversion of Archaeal Zinc-containing Ferredoxin from Sulfolobus sp. Strain 7^*

Toshio Iwasaki^§, Eiji Watanabe^¶, Daijiro Ohmori, Takeo Imai^¶, Akio Urushiyama^¶, Minoru Akiyama^¶, Yoko Hayashi-Iwasaki^**, Nathaniel J. Cosper^§§¶¶, and Robert A. Scott^§§

From the  Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan, the ^¶ Department of Chemistry, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo 171-8501, Japan, the  Department of Chemistry, Juntendo University, Inba, Chiba 270-1695, Japan, the ^** Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Tokyo 192-0392, Japan, and the ^§§ Center for Metalloenzyme Studies and Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556

Received for publication, November 15, 1999, and in revised form, May 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and ~6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and ¹H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S]¹⁺ cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ferredoxins, small iron-sulfur (FeS) proteins in archaea, serve as water-soluble electron acceptors of acyl-coenzyme A forming 2-oxoacid:ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways (1-5). The 2.0-Å resolution x-ray crystal structure (PDB¹ entry 1XER ; Ref. 6) of ferredoxin from Sulfolobus sp. strain 7 (JCM 10545; optimal growth conditions, pH 2.5-3 and 80 °C) showed the presence of an unexpected isolated zinc center, tetrahedrally coordinated by three nitrogen atoms from histidine residues in the N-terminal extension region (N1 of His¹⁶, N2 of His¹⁹, and N1 of His³⁴) and one O1 atom from Asp⁷⁶ in the FeS cluster-binding core fold (Fig. 1). A similar zinc site was also found in ferredoxin from Thermoplasma acidophilum strain HO-62 that also contained one [3Fe-4S]^1+,0 cluster, and one [4Fe-4S]^2+,1+ cluster (7). This site was analyzed by zinc K-edge x-ray absorption spectroscopy (XAS) (8) and was found to be essentially identical to the zinc site in Sulfolobus sp. ferredoxin. Thus, these unusual ferredoxins contain both the conventional FeS clusters and a structurally conserved, isolated zinc center. This new class of bacterial type ferredoxin, isolated from phylogenetically diverse members of several aerobic and thermoacidophilic archaea, are thus called "zinc-containing ferredoxins" (2, 6-10).

Another unexpected result of the crystal structure of air-oxidized Sulfolobus sp. zinc-containing ferredoxin was the presence of two [3Fe-4S] clusters beside one isolated zinc center (9) (Fig. 1). The number and type of FeS clusters in this structure are inconsistent with our previous spectroscopic analysis of the purified protein, which suggested one [3Fe-4S]^1+,0 cluster (cluster I; E_1/2 = 280 mV) and one [4Fe-4S]^2+,1+ cluster (cluster II; E_1/2 = 530 mV) (5). Other archaeal zinc-containing ferredoxins from T. acidophilum (7, 8), Sulfolobus acidocaldarius (11), and Acidianus ambivalens (formerly Desulfurolobus ambivalens) (12) also contain one [3Fe-4S] cluster and one [4Fe-4S] cluster, rather than two [3Fe-4S] clusters. The primary structure of Sulfolobus sp. ferredoxin showed the presence of seven cysteines that are arranged in two FeS cluster-binding motifs, one from the sequence, Cys⁴⁵-Leu-Ala-Asp⁴⁸-Gly-Ser-Cys⁵¹ and Cys⁹³-Pro, and the other from the sequence, Cys⁵⁵-Pro and Cys⁸³-Ile-Phe-Cys⁸⁶-Met-Ala-Cys⁸⁹ (5, 13). The latter motif provides a typical binding site for a [4Fe-4S] cluster with complete cysteinyl ligation, although Cys⁸⁶ does not serve as a ligand to the [3Fe-4S] cluster II in the crystal structure (Fig. 1).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   The 2.0-Å resolution crystal structure of the 6Fe-containing form of zinc-containing ferredoxin from Sulfolobus sp. strain 7 (PDB entry 1XER.pdb; Ref. 9), colored by the temperature factors. The metal centers (zinc, cluster I, and cluster II) and important residues are labeled, and -sheet structures are shown transparently in pink. In this structure, the region with the highest average temperature factors (blue) is in the vicinity of the [3Fe-4S] cluster II, especially around Cys86 and Met87, whereas other part mostly shows lower temperature factors (red). Of seven cysteine residues, six serve as the ligands to the two FeS clusters, whereas Cys86 in the vicinity of the [3Fe-4S] cluster II is exposed to the solvent, as in the case of Cys11 of D. gigas ferredoxin II (PDB entry, 1FXD.pdb; Ref. 17) (not shown in the figure). The figure was prepared using Insight II (Molecular Simulations Inc.).

Because FeS clusters have a remarkable facility for interconversion under protein-bound conditions (reviewed in Ref. 14), the significant discrepancy of the types of FeS clusters of Sulfolobus sp. zinc-containing ferredoxin in the crystalline and as-isolated states may represent selective degradation of the cluster II to a [3Fe-4S] form in vitro. Although the [4Fe-4S]  [3Fe-4S] cluster interconversion is well known in some bacterial type ferredoxins (14-25) and aconitase (26-29), there is no conclusive report demonstrating the selective cluster conversion at the cluster II site in bacterial type dicluster ferredoxins. The most extensive spectroscopic analyses of oxidative degradation process in dicluster ferredoxins have been conducted with Azotobacter vinelandii ferredoxin I with one [3Fe-4S] cluster and one [4Fe-4S] cluster (30-35). In this instance, it has been proposed that ferricyanide oxidation results in complete destruction of the [4Fe-4S] cluster, to produce an intermediate containing only one [3Fe-4S] cluster (cluster I), en route to complete disruptions of both clusters and ultimately to unfolded protein (30, 31)_.

We have recently found conditions where the native 7Fe form of Sulfolobus sp. ferredoxin (5) is slowly and irreversibly converted to a stable intermediate species under aerobic conditions (10). Herein, we report comparisons of EPR, resonance Raman, XAS, and ¹H nuclear magnetic resonance (NMR) analyses of the metal centers in the native 7Fe and the intermediate forms of Sulfolobus sp. zinc-containing ferredoxin. Our spectroscopic data demonstrate the oxidative degradation of cluster II to form a [3Fe-4S] cluster, yielding a 6Fe-containing intermediate of zinc-containing ferredoxin. This will be also discussed with respect to the structure and evolution of a ferredoxin core fold module.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DEAE-Sepharose Fast Flow and Sephadex G-50 gels were purchased from Amersham Pharmacia Biotech, and NMR-grade D₂O was from Wako Pure Chemicals (Tokyo, Japan). Water was purified by the Milli-Q purification system (Millipore). Other chemicals used in this study were of analytical grade.

Sulfolobus sp. strain 7 cells (JCM 10545), originally isolated from Beppu Hot Springs, Japan, were cultivated aerobically and chemoheterotrophically at pH 2.5-3 and 75-80 °C, and the 7Fe form of the archaeal ferredoxin was routinely purified as described previously (5, 10). The intermediate form of Sulfolobus sp. ferredoxin was obtained by artificial conversion at pH 5.0 as described previously (10), after removal of the unconverted 7Fe form by a DEAE-Sepharose Fast Flow column chromatography (Amersham Pharmacia Biotech) followed by a Sephadex G-50 column chromatography (Amersham Pharmacia Biotech). 2-Oxoacid:ferredoxin oxidoreductase of Sulfolobus sp. strain 7 was purified as described previously (5, 36). 2-Oxoacid:ferredoxin oxidoreductase activity was monitored with a horse heart cytochrome c reduction assay at 50 °C using purified ferredoxin an intermediate electron acceptor (2, 5). Enzymatic reduction of Sulfolobus sp. ferredoxin with the cognate 2-oxoacid:ferredoxin oxidoreductase was conducted under anaerobic conditions at 55 °C as described previously (5).

Absorption spectra were recorded using a Hitachi U3210 spectrophotometer or a Beckman DU-7400 spectrophotometer. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of purified apoferredoxin (made in distilled water) was performed by a Finnigan MAT VISION 2000 instrument at an accelerating potential of 5.0 kV, using a 2,5-dihydroxybenzoic acid matrix. Electron paramagnetic resonance (EPR) measurements were performed using a JEOL JES-FE3XG spectrometer equipped with an Air Products model LTR-3-110 Heli-Tran cryostat system and a Scientific Instruments series 5500 temperature indicator/controller. Spin concentrations were estimated by double integration, with 0.1 and 1 mM Cu-EDTA as standards. The spectral data were processed using KaleidaGraph version 3.05 (Abelbeck Software).

Low temperature resonance Raman spectra were recorded at 77 K using 488.0-nm and 457.9-nm Ar⁺ laser excitation (500 mW) as described previously (7, 37). The sample was immersed into a liquid nitrogen reservoir and the scattered light was collected at 45 degrees to the incident beam. The spectral slit width was 4 cm¹, and a multiscan averaging technique was employed.

Purified zinc-containing ferredoxins in 20 mM potassium phosphate buffer, pH 6.8, were concentrated by pressure filtration with an Amicon YM-3 membrane. Further concentration was achieved by placing the samples under a stream of dry nitrogen gas. The resultant samples (~2-3 mM), containing 30% (v/v) glycerol, were frozen in a 24 × 3 × 2-mm polycarbonate cuvette with a Mylar-tape front window for XAS studies. XAS data were collected at Stanford Synchrotron Radiation Laboratory with the SPEAR storage ring operating in a dedicated mode at 3.0 GeV (Table I), as reported previously (8). EXAFS analysis was performed with the EXAFSPAK software according to standard procedures (38). Curve-fitting analysis was performed as described previously (8, 39). Multiple scattering models, calculated using FEFF version 7.02 (40), were based on bis(acetato)-bis(imidazole)-zinc(II) (41) or tetra(imidazole) zinc(II) perchlorate (42).

¹H NMR spectra were recorded on using JEOL GSX-400 spectrometer operating at 399.78 MHz Larmor frequency. The NMR sample was prepared by exchanging the buffer of purified ferredoxin into 10 mM sodium deuterium phosphate buffer, pH 7.5 (pH was uncorrected), using an Amicon ultrafiltration device with a YM-3 membrane (final concentration, ~3 mM ferredoxin). Sweep widths of 30 KHz were used for the purified protein. ¹H spectra were recorded with either a slow repetition time (2 s) using presaturation for water suppression or with a fast repetition time (100 ms) using a super water elimination Fourier transform (super-WEFT) pulse sequence (43) with a relaxation delay (60 ms) between the  and /2 pulse sequence for water suppression. The spectra were calibrated by referencing to the residual HDO signal, assigned to a 4.74-ppm shift at 303 K. The standard JEOL software package was used for data processing.

Protein concentration of purified ferredoxins was measured as described previously (10). Metal content analyses were carried out by inductively coupled plasma atomic emission spectrometry with a Seiko SPS 1500 VR instrument at Tokyo Institute of Technology and a Jobin-Yvon JY 38S instrument at Rigaku Ltd.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The typical yield of the purified intermediate obtained from the 7Fe form was ~30% under the conditions described under "Experimental Procedures." The matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that masses [M + H]¹⁺ of these apoproteins are identical within the experimental error (data not shown). Chemical analysis by inductively coupled plasma atomic emission spectrometry suggested the iron:zinc ratio of 6.9:1.0 mol/mol in the native 7Fe form and 5.6:1.0 mol/mol in the intermediate form.

EPR Properties of the Native 7Fe and Intermediate Forms of Sulfolobus sp. Ferredoxin-- The 7Fe form of Sulfolobus sp. ferredoxin elicited a sharp g = 2.02 EPR signal (0.9-1.0 spin/mol) attributed to a [3Fe-4S]¹⁺ cluster as reported previously (5, 8) (Fig. 2, trace A). The intermediate form also elicited a sharp EPR signal at g = 2.02, detectable up to 20 K, but with different lineshapes and relaxation behavior. The microwave power saturation behavior of the EPR signal at 8 K, of the intermediate form (P_1/2 of 36 mW assuming a single component; P_1/2 of 0.7 mW and 110 mW assuming two components; open squares in Fig. 2B), was also different from that of the native 7Fe form (P_1/2 of 60 mW; closed squares in Fig. 2B). Under non-saturation conditions, the spin concentration of the g = 2.02 EPR signal of the intermediate was estimated to be ~1.7 spin/mol, indicating the presence of approximately two S = 1/2 [3Fe-4S]¹⁺ clusters. No EPR signals were detected at temperatures above 35 K, suggesting the absence of a stable radical species in the air-oxidized intermediate (cf. Refs. 30, 33, and 34).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   EPR spectra of the native 7Fe form of zinc-containing ferredoxin from Sulfolobus sp. ferredoxin and its intermediate form in the air-oxidized state (A); the power saturation curves of the oxidized protein at 8 K are shown in B. The power saturation curves were fitted with P1/2 of 36 mW assuming a single component (dashed trace) (P1/2 of 0.7 and 110 mW, assuming two components; solid trace) for the intermediate form (open squares), and P1/2 of 60 mW for the native 7Fe form (solid trace and closed squares). The samples were dissolved in 600 mM CAPS buffer, pH 9.3, and adjusted at the same concentrations. Modulation amplitude of the instrument is 0.63 millitesla, and the g values and other conditions are indicated in the figure.

Upon reduction of the native 7Fe form, the sharp g = 2.02 EPR signal attributed to that of a [3Fe-4S]¹⁺ cluster was fully reduced, thus giving rise to a very broad low field resonance at g ~ 12, which is characteristic of the reduced S = 2 [3Fe-4S]⁰ cluster (44) (Fig. 3, trace A). A rhombic EPR signal at g = 2.06, 1.94, and 1.90, attributed to the reduced S = 1/2 [4Fe-4S]¹⁺ cluster, was detected. This signal had additional wings on the high and low field sides of the main EPR signal (g = 2.11 and 1.85), resulting from magnetic interactions with the reduced S = 2 [3Fe-4S]⁰ cluster. These EPR signals could be detected up to 30 K (data not shown), and no evidence for the presence of a high multiplicity S = 3/2 [4Fe-4S]¹⁺ cluster was obtained (Fig. 3, trace A).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   EPR spectra of the native 7Fe form of zinc-containing ferredoxin from Sulfolobus sp. ferredoxin (A) and its intermediate form (B-D) in the dithionite-reduced state at pH 9.3. The samples used for the measurement are the same as in Fig. 2.

Reduction of the intermediate under the same conditions resulted in the disappearance of most of the sharp g = 2.02 EPR signal attributed to the S = 1/2 [3Fe-4S]¹⁺ clusters, and the appearance of the broad low field resonance at g = 12 characteristic of the S = 2 [3Fe-4S]⁰ cluster, as the predominant species (Fig. 3, traces B, C, and D). Several weak and minor resonances, mainly consisting of the remaining S = 1/2 [3Fe-4S]¹⁺ cluster at g = 2.02 and a very weak radical feature at g = 2 of unknown origin, were also reproducibly detected in the g ~ 2 region (<0.1 spin/mol). All these minor species existed in a substoichiometric amount (<0.1 spin/mol), indicating minor heterogeneity in the dithionite-reduced intermediate form.

Resonance Raman Spectroscopy-- Low temperature resonance Raman spectroscopy has been utilized as a probe for distinguishing between types of oxidized FeS clusters (16, 45-47). Hence, the properties of the air-oxidized FeS clusters of Sulfolobus sp. ferredoxin were investigated by resonance Raman spectroscopy (Fig. 4, A and B). Based on extensive assignments by Spiro and co-workers (45, 46), it appears that the [3Fe-4S]¹⁺ cluster of the native 7Fe form exhibits three primarily Fe-S bridging modes (260, 285, and 347 cm¹) and at least two Fe-S terminal modes (369 and 385 cm¹) (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Resonance Raman spectra at 77 K of the native 7Fe form (A and B) and the intermediate form (C and D) of Sulfolobus sp. ferredoxin recorded using 457.9-nm (A and C) and 488.0-nm (B and D) Ar+ ion laser excitation (500 mW). The weak band at 335 cm1 detected in the native 7Fe form (A and B) was not detected in the 6Fe-containing intermediate form, indicating a loss of a [4Fe-4S]2+ cluster (C and D). The spectral slit width was 4 cm1, and a multiscan averaging technique was employed. Concentration of the samples was ~5 mM.

A weak band at 335 cm¹ appears in the native 7Fe form and is assigned to the Fe-S bridging mode of a regular biological [4Fe-4S]²⁺ cluster with complete cysteinyl ligation (46) (Fig. 4A). In Pyrococcus furiosus 4Fe ferredoxin and active aconitase, which contain a [4Fe-4S] cluster coordinated by one non-cysteinyl and three cysteinyl ligands (16, 48, 49), the equivalent band was shifted to a higher frequency (16). It has been reported that the resonance Raman spectra for biological [4Fe-4S]²⁺ clusters are normally much less intense than those for biological [3Fe-4S]¹⁺ clusters (reviewed in Refs. 45 and 47). Relative intensity of the Fe-S bridging mode of a [4Fe-4S]²⁺ cluster at 335-cm¹ band in the native 7Fe form of Sulfolobus sp. ferredoxin (Fig. 4A) is very similar to that reported for Thermus thermophilus 7Fe ferredoxin (45, 47). A very weak Fe-S terminal mode at ~249 cm¹ is normally seen for [4Fe-4S]²⁺ clusters; however, the signal in this region, for the 7Fe form of Sulfolobus sp. ferredoxin, is too weak to analyze. In conjunction with other spectroscopic data reported in this paper (Figs. 2 and 3), the resonance Raman data of the native 7Fe form indicate the presence of a [4Fe-4S]²⁺ cluster, in addition to a [3Fe-4S]¹⁺ cluster.

The low temperature resonance Raman spectrum at 488.0-nm Ar⁺ ion laser excitation of the intermediate form showed that the [3Fe-4S]¹⁺ cluster also exhibited three Fe-S bridging modes at 260, 285, and 347 cm¹, and at least two Fe-S terminal modes at 369 and 385 cm¹ (Fig. 4D). Detection of these bands in both the native 7Fe (Fig. 4B) and intermediate 6Fe forms (Fig. 4D) suggests that the [3Fe-4S]¹⁺ core structure is structurally not very different in these two forms. On the other hand, the weak band primarily associated with the Fe-S bridging mode at 335 cm¹ of the [4Fe-4S]²⁺ cluster of the native 7Fe form, resonantly enhanced upon 457.9-nm Ar⁺ ion laser excitation (Fig. 4A), was not detected in the intermediate form (Fig. 4C). These results are consistent with the absence of a [4Fe-4S]²⁺ cluster in the intermediate form. It should be noted that the band at 359 cm¹, resonantly enhanced upon 457.9-nm Ar⁺ ion laser excitation (Fig. 4, A and C), was detected in both forms of Sulfolobus sp. ferredoxin, whereas the equivalent band was observed in the 7Fe, but not the 6Fe, form of Mycobacterium smegmatis ferredoxin (37, 50). This indicates that a weak band at ~359 cm¹ cannot be used diagnostically for the presence of a conventional biological [4Fe-4S]²⁺ cluster.

X-ray Absorption Spectroscopy-- The zinc K-edge x-ray absorption spectra of the purified 7Fe and 6Fe forms of Sulfolobus sp. ferredoxin are very similar (Fig. 5). The absorption edge positions (9663.2 for 7Fe, 9663.3 for 6Fe) for both samples fall at the expected energy for Zn(II) with all light elements (nitrogen or oxygen) in the coordination sphere (51, 52).² The intensity of the edge is most reminiscent of four-coordinate compounds, and the peak area of the second XANES peak is not as intense as expected for tetraimidazole coordination, nor is it as weak as seen in a ZnO₄ compound (51).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Zinc (left) and iron (right) x-ray absorption spectra of the native 7Fe- (dashed line) and the 6Fe-containing intermediate form (solid line) of Sulfolobus sp. ferredoxin.

The presence of a carboxylate ligand results in destructive interference with the EXAFS multiple-scattering contributions from outer shell atoms of histidine imidazoles. This interference can be visualized in the Fourier transform of the data as a decrease in the ~3-Å peak relative to the ~4-Å peak. Previously, we reported that the 7Fe form of Sulfolobus sp. ferredoxin was best fit assuming a Zn(imid)_3,4(COO)₁ coordination environment with a Zn-O-C bond angle of ~126° (Fit 4, Table II) (8).³ Similarly, the 6Fe form can also be fit assuming the same coordination geometry (cf. Fits 4 and 8, Table II; Fig. 6). The zinc XAS results clearly show that the zinc site found in the intermediate is an isolated center having very similar coordination environment to the native 7Fe form. The XAS-determined bond distances and bond angles of the intermediate are also in agreement with the crystallographically determined Zn-N and Zn-O bond distances (1.96 and 1.90 Å, respectively) and Zn-O-C angle (~126°) (6).

View this table: [in this window] [in a new window]   Table II EXAFS curve fitting results Ras is the metal-scatterer distance. as2 is a mean square deviation in Ras. The shift in EO for the theoretical scattering functions was optimized, but did not vary more than 1.5 eV. Numbers in square brackets were constrained to be either a multiple of the above value (as2) or to maintain a constant difference from the above value (Ras). f' is a normalized error (chi-squared):

View larger version (25K): [in this window] [in a new window]   Fig. 6.   k3-weighted zinc (left) and iron (right) EXAFS (insets, over k = 2-12.5 Å1, for zinc; k = 2-13.5 Å1, for iron) and Fourier transforms of native 7Fe (dashed line) and intermediate 6Fe (solid line) forms of Sulfolobus sp. ferredoxin.

The iron K-edge x-ray absorption spectra for the two forms of Sulfolobus sp. ferredoxin were almost identical (Fig. 5). Curve-fitting analysis of the native 7Fe form and the stable intermediate reveals the presence of a 2.25-2.26-Å Fe-S and a 2.71-2.72-Å Fe-Fe interaction (8). The best fit (by goodness-of-fit values) is obtained from calculated EXAFS for FeS₄Fe₂ (Fit 10, Table II). However, the data can also be fit assuming FeS₄Fe_2.5 (Fit 11, Table II), as expected for one [3Fe-4S] and one [4Fe-4S] cluster (8). EXAFS for the 6Fe form (Fig. 6) is best fit assuming FeS₄Fe₂ (Fit 13, Table II), as expected for two [3Fe-4S] clusters. The goodness-of-fit value is lower for FeS₄Fe₁ (Fit 12, Table II); however, the Debye-Waller factor for the Fe-Fe interaction is physically unreasonable. Similar to the 7Fe form, the data can also be fit assuming FeS₄Fe_2.5 (Fit 14, Table II). However, the Debye-Waller factors indicate that there are on average fewer (or more disordered) Fe-Fe interactions in the 6Fe form than in the 7Fe form, as would be expected given the [4Fe-4S]  [3Fe-4S] cluster conversion. It should be noted that the EXAFS analysis of the oxidatively degraded 3Fe-containing intermediate and a single crystal of the native 7Fe form of A. vinelandii ferredoxin I showed the average Fe-Fe distance of ~2.7 Å (32, 35, 53), similar to the results obtained here.

¹H NMR Spectroscopy-- ¹H NMR spectroscopy has been used to detect and assign the -protons of cysteine ligand residues coordinated to the FeS centers in some 7Fe-containing ferredoxins (54-57). Bentrop et al. (55) have performed detailed paramagnetic NMR analysis of the native 7Fe form of another zinc-containing ferredoxin from A. ambivalens, which is 95% identical to Sulfolobus sp. ferredoxin at the primary structural level (only 5 out of 103 amino acids are different) (7, 12, 58). Interestingly, the ¹H NMR spectrum of A. ambivalens ferredoxin shows eight major and eight minor hyperfine-shifted resonances, arising from its ligand protons. This unique pattern apparently results from the superposition of typical spectra from a 3Fe- and a 4Fe-containing cluster (55). This pattern has not been reported for other regular bacterial 7Fe-containing ferredoxins, all of which show only five hyperfine-shifted resonances in the downfield region (54, 56, 57, 59-62). It has been reported that eight major and eight minor hyperfine-shifted resonances likely reflect a heterogeneity of one of the two clusters of A. ambivalens ferredoxin, which does not simply result from sample impurity (55).

The overall spectral features, chemical shift values, and temperature dependences of the hyperfine-shifted resonances in the downfield ¹H NMR spectrum of native Sulfolobus sp. ferredoxin (Fig. 7, A, C, and D) are essentially identical to those of the closely related A. ambivalens ferredoxin. A. ambivalens has complete cysteinyl coordination to both the [3Fe-4S]¹⁺ cluster and [4Fe-4S]²⁺ cluster (55). Eight major signals (C, G, H, J, K, N, O, P) and eight minor signals (A, B, D, E, F, I, L, M) were detected in the downfield region. Each of these hyperfine-shifted resonances displays an anti-Curie temperature dependence, with the exception of major signals, C and H, and minor signals, D and E, which have Curie-type temperature dependences (Fig. 7, C and D). Based on the assignment of the hyperfine-shifted resonances of A. ambivalens ferredoxin (55), major signals C, H, G, and K are attributed to ligands of the [3Fe-4S]¹⁺ cluster I, and major signals J, N, O, and P to ligands of the [4Fe-4S]²⁺ cluster II (Fig. 7, A and C). The sequence specific-assignment of the cysteine resonances in A. ambivalens ferredoxin (55) also indicates that the major signal J of the native 7Fe form is attributable to the H proton of Cys⁸⁶, which is located in the [4Fe-4S] cluster II site. It should be noted that Cys⁸⁶ is located in the vicinity of the missing corner (Fe) of the cube at the [3Fe-4S] cluster II site in the x-ray crystal structure of the 6Fe form of Sulfolobus sp. ferredoxin (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   One-dimensional 1H NMR spectrum at 303 K of the native 7Fe form (A) and the partially converted sample by oxidative degradation (B) of Sulfolobus sp. zinc-containing ferredoxin. The hyperfine-shifted resonances were labeled according to the 1H NMR data of Bentrop et al. (55). The spectrum of samples in B was measured after storage of sample A in NMR tubes for 7 months under auxogenic conditions at 5 °C. Concomitant with the decrease of major signals, J, N, O, and P, several hyperfine-shifted resonances appeared at around 16.0 ppm (signal b), 21.4 ppm (not labeled), and 24.0-24.6 ppm (signal a) in sample B. The temperature dependences of the hyperfine-shifted resonances (in a chemical shift versus 1/T plot) of the major species (C), the minor species (D) of the native 7Fe form (spectrum A), and the newly developed species (E) of the partially converted sample (spectrum B) are also shown in the figure.

The overall spectral features and chemical shift values of the downfield resonances did not change when stored at 5 °C for a short period (within 1 month). However, storage of the native 7Fe form, under oxygenic conditions at 5 °C, in NMR tubes for a long term period resulted in gradual and irreversible changes in the spectrum (Fig. 7B). The relative intensities of major signals J, N, O, and P gradually decreased in 7 months, whereas the chemical shift values and relative intensities of other major signals C, H, G, and K, remained unchanged under these conditions (Fig. 7B). This process was sluggish and did not go to completion.⁴ The low temperature resonance Raman spectrum of this sample showed a decrease of the Fe-S bridging mode at 335 cm¹ attributed to the [4Fe-4S]²⁺ cluster II (Fig. 8B; see also Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Resonance Raman spectra at 77 K of the native 7Fe form (A) and the partially converted sample used for the 1H NMR measurement (B) of Sulfolobus sp. ferredoxin recorded using 457.9-nm Ar+ ion laser excitation (500 mW). The band at 335 cm1 decreased in spectrum B, as compared with the native 7Fe form (A) (cf. Fig. 4C). The spectral bandwidth was 4 cm1, and a multiscan averaging technique was employed.

Concomitant with the decrease of major peaks, J, N, O, and P, several hyperfine-shifted resonances appeared at around 16.0, 21.4, and 24.0-24.6 ppm (Fig. 7B). The chemical shift values of the new peaks at 16.0 ppm (b) and 24.0-24.6 ppm (a) are very similar to the hyperfine-shifted resonances B1 and B4 of ferricyanide-treated dicluster ferredoxin of Thermus thermophilus HB8 (60). The temperature dependence of the new peak a at 24.0-24.6 ppm in the downfield spectrum of Sulfolobus sp. ferredoxin suggested splitting into two peaks, a1 and a2, at 30 °C (Fig. 7E), as previously observed for the downfield-shifted resonances at around 24.5 ppm of the 3Fe form of P. furiosus monocluster ferredoxin (63-65). In addition, the new signal a1 at 24.0-24.6 ppm showed Curie-type temperature dependence (Fig. 7E), as observed with the major signal pair C and H and minor signal pair D and E, which are attributed to one ligand (Cys^IV) of a [3Fe-4S] cluster domain (55) (Fig. 7, C and D). The details of the sequence-specific assignments of the new hyperfine-shifted resonances are the subject of future analysis, and their correlation with the minor peaks observed in the native 7Fe form remain unclear at this stage. However, some of these resonances (such as signal a) probably belong to a [3Fe-4S] cluster domain and are in line with the increase of the EPR signal intensity at g = 2.02 (data not shown; see Fig. 2).

Taken together, these data suggest that the [4Fe-4S] cluster II in the native 7Fe form is less stable to oxidative degradation than the [3Fe-4S] cluster I, and that the [4Fe-4S]²⁺ cluster II is gradually and selectively converted to a corresponding cubane [3Fe-4S]¹⁺ cluster II, whereas the [3Fe-4S] cluster I remains essentially unchanged under the applied conditions. Further ¹H NMR analysis is underway to reveal the sequence-specific assignments of the new hyperfine-shifted resonances and the electronic structure of the two [3Fe-4S] clusters in the 6Fe-containing intermediate form.

Biochemical Properties-- Archaeal zinc-containing ferredoxins have been shown to serve as water-soluble electron acceptors of acyl-coenzyme A forming 2-oxoacid:ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways (2, 3, 5, 7, 12). Previous enzymatic reduction of the native 7Fe form by Sulfolobus sp. 2-oxoacid:ferredoxin oxidoreductase reduced only the [3Fe-4S] cluster I, whereas the [4Fe-4S] cluster II with a lower midpoint redox potential remained in the oxidized state during the steady state (5). The specific activity of the cognate 2-oxoacid:ferredoxin oxidoreductase in the presence of 4 mM 2-oxoglutarate, 50 µM coenzyme A, and 23 µg of the 6Fe-containing intermediate form at 50 °C was 55 µmol/min/mg, only slightly lower than that measured with the native 7Fe form under the same conditions (60 µmol/min/mg) (5, 36). Thus, the reactivity with the cognate oxidoreductase was not lost after the [4Fe-4S]  [3Fe-4S] cluster conversion. Enzymatic reduction of the 6Fe form with a catalytic amount of the 2-oxoacid:ferredoxin oxidoreductase (8.6 µg/ml) in the presence of 4 mM 2-oxoglutarate and 0.5 mM coenzyme A during the steady-state turnover of the oxidoreductase under anaerobic conditions at 55 °C caused bleaching of the g = 2.02 EPR signal attributed to the [3Fe-4S]¹⁺ clusters (see Fig. 3) by ~95% in 30 min, with concomitant formation of the broad low field resonance at g ~ 12, which is indicative of the S = 2 [3Fe-4S]⁰ clusters (data not shown). This indicates that the two [3Fe-4S] clusters, i.e. both clusters I and II, of the 6Fe form can be reduced enzymatically by the cognate oxidoreductase under the applied conditions, due to the change of electron distributions within the reduced ferredoxin molecule by relative redox potential upshift of the cluster II upon the selective cluster conversion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present spectroscopic investigation indicates that the initial step of oxidative degradation of the native 7Fe form of Sulfolobus sp. zinc-containing ferredoxin, at fairly acidic or neutral pH, is a selective conversion of the [4Fe-4S] cluster II to a [3Fe-4S] cluster. This degradation pathway results in a stable 6Fe-containing intermediate with one isolated zinc center and two [3Fe-4S] clusters, en route to complete protein unfolding. Although the selective interconversion of a [4Fe-4S]  [3Fe-4S] cluster at the cluster I site has been well established in some bacterial ferredoxins (14-25), this novel intermediate is unique in that its formation is triggered by selective cluster conversion at the cluster II site, rather than complete destruction of the cluster. The local metric environments of the [3Fe-4S] cluster I and isolated zinc site in the intermediate form do not change significantly as compared with those in the native 7Fe form, indicating that structural rearrangement of the whole molecule does not occur at the initial step of oxidative degradation. The ability of the intermediate to accept electrons transferred from the cognate 2-oxoacid:ferredoxin oxidoreductase is also maintained at this stage.

Correlation with the X-ray Crystal Structure of the 6Fe Form of the Sulfolobus Ferredoxin-- The spectroscopic properties of the stable intermediate form of Sulfolobus sp. ferredoxin are consistent with the structural features observed for the 2.0-Å resolution crystal structure of the 6Fe form (6, 9). In this structure, the [3Fe-4S] cluster I is coordinated by three cysteinyl ligands contributed from Cys⁴⁵, Cys⁵¹, and Cys⁹³, and the [3Fe-4S] cluster II by another three cysteinyl ligands contributed from Cys⁵⁵, Cys⁸³, and Cys⁸⁹ (9) (Fig. 1). The two [3Fe-4S] clusters are separated by a crystallographic, center-to-center distance of 12.0 Å, which is similar to that observed in some regular 7Fe- and 8Fe-containing dicluster ferredoxins. The second cysteine residue (Cys⁸⁶) in the -Cys⁸³-Xaa-Xaa-Cys⁸⁶-Xaa-Xaa-Cys⁸⁹-Xaa-Xaa-Xaa-Cys⁹³-Pro- motif is located in the vicinity of the missing corner (Fe) of the cluster II cube, and its side chain is exposed to the solvent, away from cluster II (Fig. 1). A similar observation has been reported for the non-ligating cysteinyl residue (Cys¹¹) in the D. gigas ferredoxin II structure with a single cubane [3Fe-4S] cluster (PDB entry 1FXD.pdb; Ref. 17). The equivalent residue (Cys¹³) of the closely related 4Fe-containing ferredoxin from Thermotoga maritima (PDB entry 1VJW.pdb; Ref. 66) serves as a ligand to the [4Fe-4S] cluster.

The electron density for Cys⁸⁶ is lower than that of other cysteinyl ligand residues in the crystal structure. Additionally, the average temperature factors for the region surrounding cluster II (~24 Ų, on average), especially Cys⁸⁶ and Met⁸⁷ (>30 Ų), are markedly higher than those for the region surrounding cluster I (~15 Ų) (9) (Fig. 1). These observations are consistent with the present spectroscopic analyses indicating the structural similarity of the native 7Fe form and the stable intermediate, and suggest that the selective cluster conversion at the cluster II site is a local event. In the crystal structure, the polypeptide conformation in the vicinity of the cubane [3Fe-4S] cluster II and Cys⁸⁶ was reported to be intermediate between the [3Fe-4S] and [4Fe-4S] cluster conformations (9). It is likely that the peptide backbone conformation in vicinity of Cys⁸⁶ and Met⁸⁷ may be somewhat perturbed upon formation of the 6Fe-containing intermediate by oxidative degradation.⁵

Based on these considerations, we suggest that oxidative degradation of Sulfolobus sp. zinc-containing ferredoxin is initiated at the [4Fe-4S] cluster II site. As a result of this initial degradation step, the iron atom bound to Cys⁸⁶ is released, forming a structurally interconvertible cubane [3Fe-4S] cluster II. Concomitant with the release of iron is a perturbation of the peptide backbone in the vicinity of Cys⁸⁶-Met⁸⁷ and rotation of the Cys⁸⁶ side chain to solvent, away from the cluster (see Fig. 1). At this stage, no significant structural rearrangement of the entire molecule occurs. Further sluggish oxidative degradation of the two [3Fe-4S] clusters eventually leads to protein unfolding of the ferredoxin core fold and accumulation of apoprotein (10), and may involve other short-lived intermediates not yet characterized.

Oxidative Degradation of Two FeS Clusters in Bacterial Type Dicluster Ferredoxins-- The reaction of the wild-type A. vinelandii ferredoxin I with an excess of ferricyanide is a more complicated oxidative degradation, leading to formation of an unusual intermediate with a three-electron oxidation at sulfur sites (30, 31, 33, 34). Site-directed mutagenesis has suggested that formation of the latter species requires the presence of non-ligating Cys²⁴ in the vicinity of a cluster sulfide (33). The difference between the oxidative degradation of ferredoxin from A. vinelandii and Sulfolobus sp. strain 7 is likely to reflect the differences in their cluster surroundings, i.e. the absence of the equivalent cysteine residue in the archaeal ferredoxin core fold domain (Fig. 1).

An analogous 6Fe-containing ferredoxin species has been isolated from M. smegmatis (37, 50) and similar hyperfine-shifted resonances appear in the downfield ¹H NMR spectra of ferricyanide-treated ferredoxins from M. smegmatis, Pseudomonas ovalis, and T. thermophilus (59-61). These observations lend credence to our suggestion that a common step in the oxidative degradation pathway, of regular bacterial type dicluster ferredoxins, is the formation of an intermediate containing two [3Fe-4S] clusters, as illustrated in Fig. 9. In some less stable dicluster ferredoxins, such an intermediate form could be detected only transiently. In other words, detection of this intermediate might depend on the overall structural rigidity of the ferredoxin core fold and/or the cluster surroundings.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   A, schematic illustration of the oxidative degradation of the ferredoxin core fold of a regular bacterial type 8Fe ferredoxin and its possible intermediate forms. Cysteines and prolines in two -CysI/I'-Xaa-Xaa-CysII/II'-Xaa-Xaa-CysIII/III'-Xaa-Xaa-Xaa-CysIV/IV'-Pro- motifs are labeled in different colors, and ligating CysII/II' are highlighted. Ligating cysteines are shown in Roman numerals, and non-ligating cysteines are in brackets. The number of iron atoms in the cluster I site is labeled in blue, and that in the cluster II site in pink. B, the selective degradation of cluster II of the zinc-containing 7Fe ferredoxin from Sulfolobus sp. strain 7, with a concomitant formation of a stable 6Fe-containing intermediate. In the case of Sulfolobus sp. ferredoxin, the initial step probably involves a release of iron atom from CysII' (Cys86), which then moves away from the cluster, as indicated from the three-dimensional structure of the 6Fe form (see Fig. 1). The selective conversion of a [4Fe-4S] cluster (cluster II) into a [3Fe-4S] cluster occurs without any significant effect on the cluster I and the remote zinc site (B). The native-like ordered structure of the cluster II site is maintained in a ferredoxin core fold when the protein-bound FeS cluster is present.

Structural and Evolutionary Implications-- The polypeptide backbone structure of a bacterial type ferredoxin exhibits a pseudo two-fold symmetry regardless of the number of bound FeS clusters. It has been proposed that the distorted two-fold symmetrical structure has been derived from a putative common ancestor as a result of early gene duplication event, and that the cluster I is strictly conserved in all bacterial type monocluster and dicluster ferredoxins reported so far (5, 22, 67). There is no monocluster ferredoxin with a cluster bound only to the cluster II site, implying that the two cluster binding sites in a ferredoxin core fold are evolutionary not equivalent (5, 67). The selective interconversion of a [4Fe-4S]  [3Fe-4S] cluster at the cluster I site has been well established in some bacterial ferredoxins (14-25), but this was not demonstrated at the cluster II site. Our results indicate that oxidative degradation of the cluster II site in dicluster ferredoxins probably follow the same chemistry with a general structural rule: The missing corner (Fe) of a biological [4Fe-4S] cube is associated with either replacement (e.g. Cys^II, Asp), or tilting away to solvent, of the second cysteine residue (Cys^II) in the -Cys^I-Xaa-Xaa-Cys^II-Xaa-Xaa-Cys^III-Xaa-Xaa-Xaa-Cys^IV-(Pro)- motif (see Figs. 1 and 9). Although it is currently unclear why this particular site is the most sensitive target for oxidative damage, we expect that increased stability of oxygen-labile bacterial type ferredoxins against oxidant (such as molecular oxygen or ferricyanide) could be conferred by introducing appropriate mutations to the polypeptide region in the vicinity of Cys^II.

A ferredoxin core fold of a bacterial type ferredoxin represents a [3Fe-4S]/[4Fe-4S] cluster-binding module in biological systems, and can be found in various simple and complex electron transfer proteins as a cluster-binding subunit and/or domain (14, 22). To the best of our knowledge, there are only a few instances of electron transfer proteins which have a [3Fe-4S] cluster at the cluster II site of the module. These include an unusual 7Fe-containing ferredoxin from a hyperthermophilic archaeon, Pyrobaculum islandicum (68) and the C-terminal domain of the cluster-binding subunit (subunit B) of some respiratory fumarate reductase and succinate dehydrogenase complexes (69-71). In all cases, the second cysteine residue (Cys^II) in the -Cys^I-Xaa-Xaa-Cys^II-Xaa-Xaa-Cys^III-Xaa-Xaa-Xaa-Cys^IV-(Pro)- motif is replaced by a non-cysteine residue such as Asp, Val, Ile, and Ala. In Escherichia coli respiratory fumarate complex, this might have afforded reduction of the [3Fe-4S] cluster II directly by proximal menaquinol (69). In the native 7Fe form of archaeal zinc-containing ferredoxins, the [4Fe-4S] cluster II is not reduced by the cognate 2-oxoacid:ferredoxin oxidoreductase (5, 7, 12), although the [3Fe-4S] cluster II of the 6Fe intermediate form can be reduced enzymatically by the oxidoreductase. Hence, the apparent effect of these amino acid replacement at the cluster II site is to modulate electron distributions within a reduced ferredoxin module by upshift of the relative redox potential of the cluster, because a [3Fe-4S]^1+/0 cluster generally has a higher midpoint redox potential than a [4Fe-4S]^2+/1+ cluster in biological systems (22). Nevertheless, the cluster conversion at the cluster II site by a single amino acid replacement seems to be not very favored in the course of molecular evolution of a ferredoxin core fold module, which may be related to the overall stability of the polypeptide backbone.

It is known that a ferredoxin core fold module of a bacterial type ferredoxin contains only a few secondary structural elements (5, 22) (see Fig. 1). The FeS cluster binding apparently contributes to the overall protein stability of the module, but the details of structural and thermodynamical roles of a metal cofactor in protein folding and unfolding reactions are little known (72, 73). Recent multidimensional heteronuclear NMR analysis of a partially unfolded high potential iron-sulfur protein under severe denaturing conditions indicated that its [4Fe-4S] cluster plays a decisive role in determining the resulting ordered structure, and that the backbone mobility increases with the structural indetermination (74). All the present results are also consistent with the primary role of an FeS cluster in retaining the native-like ordered structure of the ferredoxin core fold module (Figs. 1 and 9). We therefore suggest that the cluster I site of a ferredoxin core fold module is strictly conserved in biological systems not only because of the functional importance in electron transfer (5), but also the structural importance as a possible nucleation site of the folding of the module that shifts equilibrium toward a native-like ordered structure presumably by forming the Fe-S(Cys) bonds in vivo. The cluster II site is less conserved in the ferredoxin core module (5, 22, 67), but our results indicate that the native-like ordered structure of the cluster II site is maintained regardless of the type of an FeS cluster ([4Fe-4S] versus [3Fe-4S]) whenever a protein-bound cluster is present (Fig. 9).

	ACKNOWLEDGEMENTS

We thank Prof. I. Bertini (University of Florence) and Prof. G. N. La Mar (University of California, Davis) for their helpful advice for paramagnetic NMR analyses of ferredoxins, and Prof. M. K. Johnson (University of Georgia), Prof. T. Oshima (Tokyo University of Pharmacy and Life Science), and Prof. T. Nishino (Nippon Medical School) for discussion. We also thank Dr. H. Ikezawa (Finnigan MAT Instruments, Inc.) for mass measurement and Dr. N. Wakiya (Tokyo Institute of Technology) and Dr. H. Daidohji (Rigaku Ltd.) for the metal content analyses by the inductively coupled plasma atomic emission spectrometry. We acknowledge Dr. Christina M. V. Stålhandske for collecting some of the XAS data.

	FOOTNOTES

^* This work was supported in part by Grant-in-aid 11169237 for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (to T. I.) and by National Institutes of Health Grant GM 42025 (to R. A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom all correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Tel.: 81-3-3822-2131 (ext. 5216); Fax: 81-3-5685-3054; E-mail: iwasaki/biochem@om.nms.ac.jp.

Present address: Dept. of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.

^¶¶ Supported by National Science Foundation Research Training Group Award DIR 90-14281 (to the Center for Metalloenzyme Studies).

Published, JBC Papers in Press, May 24, 2000, DOI 10.1074/jbc.M909243199

² Edge position energies were calculated by determining the maxima of the first derivative of the absorption edge.

³ The number of imidazoles from this analysis is not absolute and probably depends on the exact geometry enforced on the carboxylate ligand.

⁴ In Sulfolobus sp. zinc-containing ferredoxin, the formation of the 6Fe-containing intermediate is very slow at around neutral pH, which is the main reason why the equivalent intermediate escaped detection in the previous ¹H NMR analysis of A. ambivalens ferredoxin (55). This most likely correlates with the local structural rigidity and/or the complete cysteinyl ligations of the cluster II site in Sulfolobus sp. ferredoxin (see "Discussion"). In this connection, it should be noted that the Asp¹⁴  Cys mutant of D. africanus ferredoxin III showed the presence of two [4Fe-4S]^2+,1+ clusters, and, unlike in native ferredoxin III, the [4Fe-4S]  [3Fe-4S] cluster interconversion reaction was sluggish and did not go to completion (19).

⁵ Preliminary computer modeling analysis by using a SYBYL molecular modeling software (TRIPOS Associates, Inc.) also suggested that simple rotation of the Cys⁸⁶ side chain did not allow appropriate coordination of this residue to the missing corner (Fe) of a structurally restrained biological [4Fe-4S] cluster introduced and superimposed to the [3Fe-4S] cluster II of Sulfolobus sp. ferredoxin (T. Iwasaki, K. Tomuro, Y. Hayashi-Iwasaki, and T. Oshima, unpublished results).

	ABBREVIATIONS

The abbreviations used are: PDB, Protein Data Bank; EXAFS, extended x-ray absorption fine structure; XAS, x-ray absorption spectroscopy; W, watt(s); CAPS, 3-(cyclohexylamino)propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES