Structural Conservation of the Isolated Zinc Site in Archaeal Zinc-containing Ferredoxins as Revealed by X-ray Absorption Spectroscopic Analysis and Its Evolutionary Implications* 210

The zfx gene encoding a zinc-containing ferredoxin from Thermoplasma acidophilumstrain HO-62 was cloned and sequenced. It is located upstream of two genes encoding an archaeal homolog of nascent polypeptide-associated complex α subunit and a tRNA nucleotidyltransferase. This gene organization is not conserved in several euryarchaeoteal genomes. The multiple sequence alignments of the zfx gene product suggest significant sequence similarity of the ferredoxin core fold to that of a low potential 8Fe-containing dicluster ferredoxin without a zinc center. The tightly bound zinc site of zinc-containing ferredoxins from two phylogenetically distantly related Archaea, T. acidophilum HO-62 and Sulfolobus sp. strain 7, was further investigated by x-ray absorption spectroscopy. The zinc K-edge x-ray absorption spectra of both archaeal ferredoxins are strikingly similar, demonstrating that the same zinc site is found in T. acidophilum ferredoxin as in Sulfolobus sp. ferredoxin, which suggests the structural conservation of isolated zinc binding sites among archaeal zinc-containing ferredoxins. The sequence and spectroscopic data provide the common structural features of the archaeal zinc-containing ferredoxin family.

□ S The on-line version of this article (available at http://www.jbc.org) contains Tables S1-S3.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number AB023294.
§ Supported by a National Science Foundation Research Training Group Award to the Center for Metalloenzyme Studies (DIR 90-14281).
tion (8,14) and the other being the membrane-bound aerobic respiratory chain containing multiple b-and d-type cytochromes (21). 2 The pioneering work by Kerscher and co-workers (8) has shown that T. acidophilum strain DSM 1728 contains a bacterial-type ferredoxin functioning as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase. The amino acid sequence of this ferredoxin was previously determined by Edman degradation of proteolytically generated peptides (10).
Recently, we purified the functionally equivalent ferredoxin from T. acidophilum strain HO-62 (20). Through chemical analysis, electron paramagnetic resonance (EPR) and low temperature resonance Raman spectroscopy, it was demonstrated that the ferredoxin contains one [3Fe-4S] 1ϩ,0 cluster, one [4Fe-4S] 2ϩ,1ϩ cluster, and one tightly bound zinc center (14), thus indicating the existence of "zinc-containing ferredoxins" among phylogenetically diverse members of several thermoacidophilic Archaea (14). Although the presence of a tightly bound zinc center is one of the most unique properties of the archaeal zinc-containing ferredoxins, the structural details of the zinc site have been characterized only for ferredoxin from Sulfolobus sp. strain 7, which was analyzed by x-ray diffraction (16).
X-ray absorption spectroscopy (XAS) is ideally suited for the investigation of the metric structural environment of specific metal sites in biomolecules (22). Herein, we report the XAS analysis of zinc-containing ferredoxins from these two phylogenetically distantly related Archaea, Thermoplasma acidophilum strain HO-62 and Sulfolobus sp. strain 7 (6,14,15), to characterize the structural properties of the zinc and iron coordination environments. We also report cloning and sequencing of the zfx gene encoding zinc-containing ferredoxin of T. acidophilum strain HO-62 (zfx for zinc-containing ferredoxin) and its flanking regions, to clarify its gene organization and the distribution of zinc-containing ferredoxin homologs in thermophilic organisms. The gene sequence and spectroscopic data provide the basis for comparison of the structural features among the archaeal zinc-containing ferredoxin family. EXPERIMENTAL PROCEDURES DEAE-Sephacel, DEAE-Sepharose Fast Flow, and Sephadex G-50 were purchased from Amersham Pharmacia Biotech. Water was purified by the Milli-Q purification system (Millipore). Other chemicals used in this study were purchased commercially and were of analytical grade.
Thermoplasma acidophilum strain HO-62 cells, originally isolated from hot sulfur springs at Ohwakudani solfataric field in Hakone, Japan, were routinely cultivated at pH 1.8 and at 56°C in 10-and 30-liter acid-resistant fermenters as described by Yasuda et al. (20), and zinc-containing ferredoxin was purified as described previously (14). Sulfolobus sp. strain 7 cells, originally isolated from Beppu Hot Springs, Japan, were cultivated aerobically and chemoheterotrophically at pH 2.5-3 and 75-80°C (23), and the 7Fe form of the cognate ferredoxin was purified as described previously (6,15).
Escherichia coli strain DH5␣, used for cloning, was grown in LB or TB medium, with 50 mg/ml ampicillin when required. Plasmids pGEMT and pGEM3Zf(ϩ) (Promega) were used for cloning and sequencing. DNA was manipulated by standard procedures (24).
The N-terminal 15 amino acid residues of T. acidophilum HO-62 ferredoxin (VKLEELDFKPKPIDE) (14) have been confirmed in the previous work to be identical to the amino acid sequence of a different strain (DSM 1728) of T. acidophilum determined by Edman degradation of proteolytically generated peptides (accession number P00218) (10). A DNA fragment encoding the zfx gene was obtained by PCR from template genomic DNA of T. acidophilum strain HO-62, using the following two oligonucleotide primers: TFP1 (corresponding to the Nterminal KPKPIDEH sequence (10,14) The resultant PCR product with expected length (ϳ370 bp) was amplified, subcloned into pGEMT vector, and sequenced with the vector-specific T7 and SP6 primers. PCR was then performed using a set of the TFP1/TFP2 and SP6/T7 primers, on a template genomic library generated by the ligation of BamHI-digested T. acidophilum genomic DNA and pGEM3Zf(ϩ). The resultant PCR products were size-fractionated on an agarose gel, extracted, subcloned into pGEMT vectors, and sequenced with primers designed from nucleotide sequence of the initial genomic PCR product. Finally, a genomic fragment was amplified using PCR primers corresponding to the 5Ј-and 3Ј-untranslated regions resulting in an intact zfx gene.
The sequence determination was performed by Sanger dideoxy sequencing with an automated DNA sequencer, ABI model 373A (Applied Biosystems Inc.). The DNA sequence was processed using the DNASIS version 3.6 software (Hitachi Software Engineering Co., Ltd.).
Data base searches were performed with BEAUTY and BLAST network services (25). Multiple sequence alignments were performed using a CLUSTAL X graphical interface (26) with minor manual adjustments.
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 (ϳ2-3 mM), containing 30% (v/v) glycerol, were frozen in a 24 ϫ 3 ϫ 2-mm polycarbonate cuvette with a Mylar-tape front window for XAS studies.
Absorption spectra were recorded with a Hitachi U-3210 spectrophotometer equipped with a thermoelectric cell holder. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) 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. EPR measurements were performed using a JEOL JEX-RE1X spectrometer equipped with an Air Products model LTR-3 Heli-Tran cryostat system and a Scientific Instruments series 5500 temperature indicator/controller. The spectral data were processed using KaleidaGraph version 3.05 (Abelbeck Software).

Sequence Analysis of the zfx Gene and Flanking Regions-
The zfx gene utilizes a translational start codon, GTG (posi-2 T. Iwasaki, unpublished results. tions 121-123, Fig. 1), and the corresponding valine residue is absent in zinc-containing ferredoxin isolated from the T. acidophilum HO-62 cells (Fig. 1), indicating post-translational modification. The single open reading frame encodes a protein with a deduced molecular mass of 15,955 Da (excluding the initial residue), which is in agreement with the average mass [M ϩ H] 1ϩ of 15,961 Da (estimated error, Ϯ 10 Da) for purified apoferredoxin by MALDI-TOF mass spectrometry. The zfx gene sequence predicts an amino acid sequence containing the three consensus histidine residues, His 30 , His 33 , and His 57 , and a remote Asp 116 (doubly-underlined in Fig. 1A). The equivalent residues in Sulfolobus sp. ferredoxin (Fig. 2) serve as ligands to the isolated zinc center (14). The deduced amino acid sequence is essentially identical to the reported sequence of T. acidophilum DSM 1728 ferredoxin determined by Edman degradation of proteolytically generated peptides (accession number P00218) (10). The two discrepancies, Glu l0l and Ala 105 , located in the central loop region (underlined residues in Fig. 1), most likely reflects the difference in strains used (strain HO-62 versus DSM 1728).
Similarity searches against available data bases (GenEMBL, PIR, and SWISS-PROT) indicate a high sequence homology of the zfx gene product with other zinc-containing ferredoxins of several fast-clock crenarchaeotes (Sulfolobales, Fig. 2), which are distantly related to the euryarchaeote T. acidophilum on the basis of the universal 16 S rRNA sequence tree (2,3,19). On the other hand, no zfx gene homolog with the consensus N-terminal extension sequence could be identified in the genomes of hyperthermophilic euryarchaeotes such as Methanococcus jannaschii (31), Methanobacterium thermoautotrophicum (32), Pyrococcus horikoshii (shinkaj) (33), Archaeoglobus fulgidus (34), and a hyperthermophilic bacterium Aquifex ae- Underlined nucleic acids represent the putative Box A, ribosome binding site, and terminating structures (term). The stop codon is over-and underlined. The predicted amino acid sequence is shown below the nucleotide sequence in the one-letter code. Amino acid residues are numbered beginning with the valine, the putative first amino acid residue of the translation product that is removed post-translationally. Underlined residues were previously determined by N-terminal sequencing (14). The probable ligand residues to an isolated zinc center of Zfx (dotted and underlined residues), and those to the two FeS clusters (dotted) are illustrated. Two other cysteine residues conserved in the zfx gene product are also shown (bold residues). The 3Ј half of the cca gene, which is not included in the 1684-bp BamHI-digested DNA fragment, was not sequenced in this study. olicus (35) by either amino acid or nucleotide sequence similarity searches (data not shown). Clearly, distribution of zinccontaining ferredoxins in hyperthermophilic and extremely thermophilic organisms is limited even in the archaeal domain.
A promoter-like element (box A) (36) was found immediately upstream of the zfx gene at positions 81-86 (Fig. 1), and a putative ribosome binding sequence (5Ј-GGTGAG-3Ј) complementary to the 3Ј end of the 16 S rRNA (19) at positions 109 -114 (underlined in Fig. 1). Because the zfx gene product is abundantly produced in T. acidophilum (8,14), the proximal promoter region of the zfx gene might be useful to express a foreign gene efficiently in this euryarchaeote. A T-rich terminator-like element (37) was found shortly after the stop codon at positions 565-573 (underlined in Fig. 1). Apparently, the zfx gene of T. acidophilum strain HO-62 does not have an operonic structure.
Two other open reading frames were found shortly after the zfx gene (Fig. 1). The first structural gene, orf1, encodes a 13.9-kDa protein with a relatively high methionine content in the N-terminal region. The Orf1 protein is strictly conserved in several thermophilic Archaea (as unknown open reading frame in Refs. [31][32][33][34], and has a domain weakly homologous to that of yeast GAL4 enhancer protein, EGD2, and mammalian nascent polypeptide-associated complex ␣ subunit (␣-NAC) (38 -42) (Fig. 3A). Mammalian ␣-NAC is a constituent of the heterodimeric nascent polypeptide-associated complex, whose heterodimerization partner has been identified as the transcription factor BTF3b (38), and has been suggested to serve as a transcriptional coactivator (41). Nascent polypeptide-associated complex is involved in ensuring signal-sequence-specific protein sorting and translocation, and is proposed to contribute to the fidelity of the recognition by modulating interactions that occur between the ribosome-nascent chain complex, the signal recognition particle and the endoplasmic reticulum membrane (38 -40, 42-44). The similarity of the Orf1 protein of T. acidophilum to eucaryal ␣-NAC homologs suggests that the archaeal protein might also serve as a putative transcriptional coactivator.
The second gene, cca, was found immediately downstream of orf1, and was partially sequenced in this study (Fig. 1). It predicts the N-terminal half of a T. acidophilum homolog of class I tRNA nucleotidyltransferase (Fig. 3B), which repairs the 3Ј-terminal CCA sequence of all tRNAs (45,46). Interestingly, the archaeal tRNA nucleotidyltransferases are similar to eucaryal poly(A) polymerases and DNA polymerase ␤, but distantly related to either the bacterial or eucaryal CCA-adding enzymes (45)(46)(47). The unique feature of the cca gene of T. acidophilum is its one-base pair overlap with the orf1 gene, implying an operonic structure; this gene organization is not observed for other hyperthermophilic euryarchaeotes with known genome sequences (31-34) (data not shown). The two structural genes downstream of the zfx gene are likely involved in translation or tRNA modification system, and apparently unrelated to the zfx gene, which is involved in cytoplasmic electron transport.
Zinc K-edge XAS Analysis-The zinc K-edge x-ray absorption spectra of the 7Fe form of zinc-containing ferredoxins purified from the two phylogenetically distantly related Archaea, T. acidophilum strain HO-62 and Sulfolobus sp. strain 7, are very similar (Fig. 4, trace a). The absorption edge position (9663.3 for T. acidophilum; 9663.2 for Sulfolobus) for both samples fall at the expected energy for Zn(II) with all light elements (nitrogen or oxygen) in the coordination sphere (48,49). 3 The intensity of the edge is most reminiscent of four-coordinate compounds and the peak area of the second XANES peak is not as 3 Edge position energies were calculated by determining the maxima of the first derivative of the absorption edge. intense as expected for tetra-imidazole coordination, nor is it as weak as seen in a ZnO 4 compound (48).
Curve-fitting analyses of zinc EXAFS of each of the two archaeal zinc-containing ferredoxins suggest the presence of three or four imidazoles. However, such Zn(imid) 3,4 (N,O) 1 fits simulate Fourier transform (FT) peaks of about the same height at 3 and 4 Å, while the observed data have a much larger FT peak at 4 Å (Fig. 5, traces a and b). This suggests that some other scatterer interferes destructively with the ϳ3-Å imidazole contribution, resulting in an absence of FT intensity. This interference can be modeled with a carboxylate group, in which the average Zn-N and Zn-O bond distances are 2.01 and 1.90 Å, respectively. The data were modeled with a Zn-O-C angle of either ϳ180°(data not shown) or ϳ126°(Fits 3 and 4 and 7 and 8, Table II), the two most common conformations found for zinc-carboxylate coordination in the Cambridge Structural Data base. The latter provides better fits of the data. Thus, the zinc K-edge EXAFS spectra of both archaeal zinc-containing ferredoxins can be best fit, assuming a Zn(imid) 3,4 (COO Ϫ ) 1 coordination environment (Fig. 5, traces a and b). 4 The zinc XAS results clearly show that the zinc site found in the zfx gene product of T. acidophilum strain HO-62 is very similar to that of Sulfolobus sp. ferredoxin. The XAS-determined bond distances and bond angles 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 (ϳ126°) (16). 4 The number of imidazoles from this analysis is not absolute and probably depends on the exact geometry enforced on the carboxylate ligand. Iron K-edge XAS Analysis-The iron K-edge x-ray absorption spectra for zinc-containing ferredoxin from T. acidophilum strain HO-62 are almost identical to that from Sulfolobus sp. strain 7 (Fig. 4, trace b). The integrated peak area (0.206 eV for T. acidophilum and 0.289 eV for Sulfolobus), for the 1 s 3 3 d transition at ϳ7113 eV, falls in the range expected for tetrahedral compounds (50 -52).
Curve-fitting analysis of both archaeal ferredoxins reveals the presence of a 2.25-2.26 Å Fe-S and a 2.71-2.72 Å Fe-Fe interaction. The best fit (by goodness-of-fit values) is obtained from calculated EXAFS for FeS 4 Fe 2 (Fits 9 and 11, Table II; Fig. 5, traces c and d). However, the data can also be fit assuming FeS 4 Fe 2.5 (Fits 10 and 12, Table II), as expected for one 3Fe and one 4Fe cluster.
EPR Spectroscopy-The air-oxidized form of both ferredoxins (Sulfolobus sp. strain 7 and T. acidophilum strain HO-62) elicited the sharp g ϭ 2.02 EPR signals with slightly different lineshapes (0.9 -1.0 spin/mol), which are attributable to a [3Fe-4S] 1ϩ cluster as reported previously (6,14) (Fig. 6, A and C). Upon reduction of these ferredoxins by excess dithionite under anaerobic conditions, the sharp g ϭ 2.02 EPR signals disappeared, and a broad low field resonance at g ϭ 12 appeared; this signal is characteristic of the reduced S ϭ 2 [3Fe-4S] 0 cluster (data not shown). In addition, rhombic EPR signals at g ϭ 2.06, 1.94, and 1.88 (Fig. 6B) and g ϭ 2.06, 1.94, and 1.90 (Fig. 6D), both attributed to a reduced S ϭ 1/2 [4Fe-4S] 1ϩ cluster, were detected up to 30 K for T. acidophilum and Sulfolobus sp. ferredoxins, respectively, together with additional wings on the high and low field sides of the main EPR signals due to magnetic interactions with the reduced S ϭ 2 [3Fe-4S] 0 cluster (Fig. 6, B and D).
Taken together, the XAS and EPR results indicate that the two archaeal zinc-containing ferredoxins contain one [3Fe-4S] 1ϩ,0 cluster and one [4Fe-4S] 2ϩ,1ϩ cluster, and that the average iron environments are nearly identical in the two proteins (Figs. 5 and 6 and Table II). The zfx gene product of T. acidophilum contains three cysteine residues arranged in a Cys 67 -Cys 68 -Ile-Ala-Asp 7l -Gly-Ala-Cys 74 , and remote Cys 133 -Pro motif, which could serve as ligands to a [3Fe-4S] cluster, and four cysteine residues in another motif, Cys 123 -Ile-Phe-Cys 126 -Met-Ala-Cys 129 , and remote Cys 78 -Pro, which are likely ligands to a [4Fe-4S] cluster (dotted cysteines in Fig. 1). The same spacing of consensus cysteine residues was found in other zinc-containing ferredoxin sequences (6,10,11,13,53), and was proposed to be attributed to the similarity of the pattern of hyperfine-shifted resonances of 1 H-NMR spectra of the 7Fe form of zinc-containing ferredoxins 5 to those of the 3Fe-, 4Fe-, and 8Fe-containing ferredoxins (53,54). In the Azotobactertype 7Fe-containing ferredoxins with a long C-terminal region, the cysteine ligand residues are arranged more asymmetrically due to the insertion of a short amino acid sequence stretch at the cluster binding motif (54 -58). The zfx sequence also shows the presence of two additional cysteine residues, Cys 66 and Cys 115 (bold residues in Fig. 1), which are not present in the Sulfolobus ferredoxin sequence (Fig. 2), and hence most likely do not serve as ligands to the clusters. DISCUSSION The sequence and spectroscopic data reported herein provide detailed structural information of the metal binding sites in T. acidophilum zinc-containing ferredoxin. The tightly bound zinc atom of archaeal zinc-containing ferredoxins constitutes an isolated and structurally conserved zinc center. The zinc is tetrahedrally coordinated with (most likely) three histidine imidazoles and one carboxylate, with average Zn-N and Zn-O bond distances of 2.01 and 1.90 Å, respectively. The sequence comparisons suggest that the three conserved histidine residues in the N-terminal extension region and one conserved aspartate in the ferredoxin core fold (Fig. 2) serve as ligands to the zinc. The similarity search for zinc-containing ferredoxin  homologs with these consensus sequence motifs against nucleotide and amino acid sequence data bases indicated their limited distribution among hyperthermophilic organisms, even within the archaeal domain (Fig. 2). This implies that early zinc-containing ferredoxins might have appeared shortly after divergence of the early Archaea, which is also in line with previous phylogenetic analysis (14).
The overall protein fold of archaeal zinc-containing ferredoxins is largely asymmetric due to the presence of a long Nterminal extension and the insertion of central loop region, as  a R as is the metal-scatterer distance. as 2 is a mean square deviation in R as . The shift in E 0 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 ( as 2 ) or to maintain a constant difference from the above value (R as ). f Ј is a normalized error (chi-squared).
compared with those of regular bacterial-type ferredoxins (Fig.  2). However, close inspection of the ferredoxin core fold suggests the strict conservation of a pseudo-two-fold symmetry with respect to the local two FeS cluster binding sites. Thus, despite the presence of one [3Fe-4S ] 1ϩ,0 cluster and one [4Fe-4S] 2ϩ,1ϩ cluster in purified proteins (6,14,15) (Fig. 2), the distribution of the conserved cysteine ligand residues in archaeal zinc-containing ferredoxins is similar to those of regular 8Fe-containing dicluster ferredoxins, except for the presence of an aspartate residue (Asp 71 in T. acidophilum ferredoxin) in place of cysteine (Fig. 2). In fact, the ferredoxin core-fold of archaeal zinc-containing ferredoxins exhibited 55-65% homology to various PsaC proteins (also called F A /F B proteins) from some phototrophic organisms and a PsaC homolog of a hyperthermophilic euryarchaeote M. jannaschii (MJ 1302; Ref. 31) (Fig. 2). PsaC is a 8Fe ferredoxin homolog found as a part of photosystem I and carries two [4Fe-4S] 2ϩ,1ϩ clusters, namely centers F A and F B , which serve as an electron donor to another FeS center, F x (59 -62). The redox potentials of the centers F A and F B of PsaC are both well below Ϫ500 mV (59), as in the cases reported for a lower potential [4Fe-4S] 2ϩ,1ϩ cluster (cluster II) of archaeal zinc-containing ferredoxins (6,12,63). Interestingly, PsaC and its archaeal analog contain a central loop region as found in archaeal zinc-containing ferredoxins, but lack the N-terminal histidine-rich stretch that contains the zinc site (Fig. 2). Because a zfx gene homolog with the consensus histidine-rich motif in the N-terminal extension region has not been found in any of the genome sequences available for aerobic and anaerobic hyperthermophiles (31)(32)(33)(34)(35), it seems plausible to postulate that early zinc-containing ferredoxins might have evolved as an 8Fe-containing low potential twoelectron carrier similar to the PsaC homolog, to which the N-terminal extension and central loop regions were attached in the later stage of molecular evolution, presumably shortly after divergence of the archaeal domain. This putative evolutionary scheme seems to be in line with the physiological function of zinc-containing ferredoxins of thermoacidophilic Archaea, serving as an electron acceptor of 2-oxoacid:ferredoxin oxidoreductases as do hyperthermophile monocluster ferredoxins without the zinc center (64 -66).
The zfx gene homologs apparently exhibit limited distribution in the archaeal domain, and have been found exclusively from the aerobic and thermoacidophilic Archaea so far (14). In thermophilic euryarchaeotes, the zfx gene product has been found only in the Thermoplasmales, an unexpected result based on the universal 16 S rRNA-based sequence tree (2,3). Analogous observation has been reported for the functionally equivalent ferredoxins of extremely halophilic and aerobic euryarchaeotes (4, 67), which contain a single plant-type [2Fe-2S] cluster and exhibit the amino acid and base sequence similarity to those of the extremely halophilic cyanobacteria (68).
In the aerobic and thermoacidophilic Archaea, the intracellular pH is maintained at pH 5.5-6.5, by the membrane-bound aerobic respiratory system operating at high temperature (23, 69 -71), implying that the cytoplasmic FeS proteins should be protected against long term exposure to the microaerobic and fairly acidic conditions during cell growth. The structurally conserved isolated zinc site of archaeal zinc-containing ferredoxins allows tight binding of the extra extension regions to one side of the ferredoxin core fold, thereby possibly providing a means to protect against gradual degradation of the bound FeS clusters under physiological conditions.