Báo cáo khoa học: High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin Linear clusters form at high pH on polypeptide unfolding doc

High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin Linear clusters form at high pH on polypeptide unfolding Susanne Griffin 1 , Catherine L. Higgins 1 , Tewfik Soulimane 2 and Pernilla Wittung-Stafshede 1 1 Department of Chemistry, Tulane University, New Orleans, LA, USA; 2 Paul Scherrer Institute, Structural Biology, Villigen, Switzerland To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 M (pH 7, 20 °C), and the thermal midpoint is above boiling, at 114 °C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear tran- siently, resulting from rearrangement of the cubic clusters into linear three-iron species. A range of iron–sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned. Keywords: ferredoxin; linear iron–sulfur cluster; protein unfolding; thermostability; Thermus thermophilus. Many proteins require the binding of cofactors to perform their biological activity. It has been demonstrated in vitro that many proteins retain interactions with their cofactors after polypeptide unfolding [1–6]. Therefore, it is possible that cofactors bind to their corresponding polypeptides before or during folding in vivo. Cofactors most often stabilize the native states of the proteins with which they interact [1–6]. However, the manner in which cofactors affect polypeptide folding and unfolding pathways remains poorly understood. Iron–sulfur ([Fe-S]) clusters represent one of nature’s simplest, functionally versatile, and perhaps most ancient cofactors [7]. The [Fe-S] clusters, which have 2, 3 or 4 irons, are usually attached to their protein partners by four cysteine thiol ligands [7–9]. Proteins that contain one or more [Fe-S] clusters represent a large class of structurally and functionally diverse proteins that are essential players in the life-sustaining processes of respir- ation, nitrogen fixation, and photosynthesis. In these proteins, the [Fe-S] clusters participate as agents of electron transfer, substrate activation, catalysis, and environmental sensing [7,10]. Most [Fe-S] proteins have low reduction potential and are known as ferredoxins. Given the struc- tural simplicity of [Fe-S] clusters and the participation of ferredoxins in so many metabolic processes, it is somewhat surprising that the pathways for biological formation of [Fe-S] clusters and their incorporation into proteins are only now beginning to emerge [7]. The origin of protein thermostability is still an unsolved problem, and its understanding presents a great intellectual challenge to scientists, not to mention its potentially enormous biotechnological impact. Proteins from thermo- philic organisms offer a unique opportunity to study the determinants of thermostability [11,12]. Although these proteins are often very similar in sequence and structure to their mesophilic homologues (this is true also for mesophilic and thermophilic ferredoxins), they are much more resistant to thermal denaturation and inactivation. In the case of thermostable ferredoxins, it is not clear if subtle features around the [Fe-S] cluster site contribute to the additional stability or if higher stability is a result of polypeptide properties only [13]. Earlier efforts to determine the origin of thermostability in monomeric proteins (most often without cofactors) have led to several hypotheses, such as stabiliza- tion by an increased number of ionic interactions, an increased extent of hydrophobic-surface burial, an increased number of prolines, and smaller surface loops [12]. Although evidence for these and other modes of stabiliza- tion can be found in specific examples, none applies to all or even most thermostable proteins. If there are general rules for how thermophilic proteins attain their stability, then it is clear that they do not lie exclusively in individual inter- actions but may be based on properties of the whole molecule [14]. In this investigation, we focus on the seven-iron (one [4Fe-4S] 2+/1+ and one [3Fe-4S] +1/ ° cluster) ferredoxin from Thermus thermophilus (hereafter called FdTt). T. ther- mophilus is a Gram-negative aerobic bacterium found in hot springs, thermal vents, and thermal spas. It grows at temperatures of 50–82 °C, with optimum growth at 65–72 °C [15]. Its seven-iron ferredoxin, FdTt, is a small single-chain, single-domain protein with 78 residues. The Correspondence to P. Wittung-Stafshede, Department of Chemistry, Tulane University, 6823 St Charles Avenue, New Orleans, LA 70118, USA. Fax: + 1 504 865 5596, Tel.: + 1 504 862 8943, E-mail: pernilla@tulane.edu Abbreviations: FdAv, ferredoxin from Azotobacter vinelandii;FdTt, ferredoxin from Thermus thermophilus; GdnHCl, guanidine hydro- chloride; T m , midpoint temperature of thermal unfolding transition. (Received 2 July 2003, revised 23 September 2003, accepted 7 October 2003) Eur. J. Biochem. 270, 4736–4743 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03873.x crystal structure of FdTt was recently solved at 1.64 A ˚ resolution [16], but there has been no in vitro characteriza- tion of the protein. FdTt has a (bab) 2 core structure enveloping the two [Fe-S] clusters with an additional C-terminal a-helix further away from the clusters (Fig. 1). According to the crystal structure of FdTt, the improved polar and hydrophobic interactions lead to extensive cross- linking of the secondary-structure elements (compared with mesophilic ferredoxins), which is believed to result in high stability. It appears that most of the stabilizing features of FdTt are found in the vicinity of the [3Fe-4S] cluster, which is the cluster that has functional importance [16]. We here report a detailed biophysical characterization of the stability of FdTt to thermal and chemical perturbation in solution in vitro. Interestingly, the cubic clusters in FdTt rearrange into new, linear three-iron species on polypeptide unfolding at high pH. Materials and methods Materials The chemical denaturant guanidine hydrochloride (GdnHCl) was of highest purity. All chemicals were purchased from Sigma. The FdTt protein was purified as previously described [16]. Chemically induced unfolding GdnHCl was used to promote FdTt unfolding at different pH values (2.5, 4, 7, 10 and 10.5) at 20 °C. An FdTt concentration of 20 l M was used unless specified otherwise. Buffers of 25 m M concentration were used unless otherwise specified. Samples were incubated (at 20 °C) for various lengths of time from 5 min to 120 h before spectroscopic measurements were taken. Unfolding was monitored by far-UV CD (200–300 nm) on a JASCO-810 instrument (1 mm cell), by visible absorption (250–700 nm) on Cary-50 and Cary-100 spectrophotometers (1 cm cell), and by tryptophan emission (300–450 nm, excitation at 280 nm) on a Cary Eclipse instrument. For each set of conditions, the transition midpoint ([GdnHCl] 1/2 ) was obtained by direct inspection of the data. Different buffers were used for experiments at different pH values: glycine/HCl was used for pH 2.5, citric acid/sodium phosphate was used for pH 4, phosphate was used for pH 7, and KCl/NaOH was used for pH 10 and 10.5. EPR experiments were performed with liquid nitrogen at 110 K on a Bruker EMX instrument (A. Tsai, University of Texas Medical School, Houston, TX, USA) using 10 mW microwave power and 9.3 GHz microwave frequency. The EPR samples were 100 l M FdTt in buffer, pH 10.5 (folded protein) and 100 l M FdTt incubated for 15 min in 6.5 M GdnHCl, pH 10.5, 20 °C (unfolded protein with linear [Fe-S] cluster). Thermally induced unfolding Thermally induced unfolding of FdTt was monitored by visible absorption, fluorescence, and far-UV CD methods. To probe the reaction by cluster integrity, the absorption at 408 nm was monitored (for FdTt samples with different pH and GdnHCl conditions) as a function of temperature. The signal from 20 l M FdTt was recorded on increasing the temperature at rates of 0.5, 0.25, or 0.125 °C per minute (one data point collected per second; from 25 °Cto95°C). At the end of each experiment, the temperature was decreased to 25 °C, and a full absorption spectrum was taken to check for refolding. The midpoint of transition (T m ) at each pH and GdnHCl concentration was deter- mined by direct inspection of the absorption vs. temperature data. T m values obtained for FdTt in the presence of different GdnHCl concentrations (but the same pH and scan rate) were used to linearly extrapolate to 0 M GdnHCl, to obtain the T m for FdTt without denaturant at each pH (and each scan rate). Next the extrapolated T m values for Fig. 1. Ribbon diagram of FdTt. Protein data bank file 1H98. The iron–sulfur clusters (iron space-filled red, sulfur space-filled yellow) and secondary-structure elements (a-helices in red, b-strands in gold, random coil in white) are highlighted. Ó FEBS 2003 Stability of T. thermophilus seven-iron ferredoxin (Eur. J. Biochem. 270) 4737 0 M GdnHCl obtained at the three different scan rates were plotted as a function of 1/(scan rate) to yield the T m for FdTt at that pH at infinite scan rate, i.e. where 1/(scan rate) ¼ 0. The T m values obtained by monitoring visible absorption were compared with those derived from far-UV CD (monitored at 220 nm) and tryptophan emission experi- ments (emission at 357 nm; excitation at 280 nm). In the case of the thermal far-UV CD-monitored experiments, we used an OLIS spectropolarimeter with a digitally controlled water bath (Julabo, Allentown, PA, USA); the approximate scan rate was 0.5 °CÆmin )1 . Results Chemically induced FdTt unfolding Folded FdTt has characteristic visible absorption at 408 nm resulting from the intact [Fe-S] clusters which disappears as the protein unfolds (Fig. 2A). FdTt has one tryptophan at position 64, and tyrosines at positions 33, 55 and 67 in the primary structure [16]. Folded FdTt shows very little tryptophan fluorescence because of energy transfer to the iron–sulfur clusters. As the protein unfolds, the tryptophan emission (at  350 nm) increases dramatically because of cluster and tryptophan separation and presumably cluster decomposition (Fig. 2B). Folded FdTt has positive CD absorption  230 nm, arising from the tryptophan and tyrosine contribution, and a negative CD feature at 220 nm, characteristic of the presence of secondary structure. Both CD bands lose intensity as the protein unfolds (Fig. 2C), and the CD spectrum on unfolding resembles that of a random-coil polypeptide. Taken together, these spectro- scopic techniques can be used to probe the unfolding reaction of FdTt via cluster integrity (visible absorption), cluster–tryptophan distance (emission), and secondary structure (far-UV CD) (Fig. 2). To probe the unfolding mechanism for FdTt, visible absorption, fluorescence, and far-UV CD probes were monitored as a function of time after the protein had been mixed with a high concentration of the chemical denaturant GdnHCl (7.9 M GdnHCl final concentration, 20 °C). Despite the high denaturant concentration, more than 6 h were required for the signals to reach their endpoints at pH 7(t 1/2 ¼ 50 ± 10 min). At pH 2.5, the kinetics for the same reaction were faster (t 1/2 ¼ 10 ± 5 min). Identical (within error) kinetic traces were observed regardless of far- UV CD; 408-nm absorption or fluorescence signals were used as the detection method (absorption and CD changes shown in Fig. 3) for each condition. This observation suggests that FdTt unfolding is a single process (at pH 2.5 and 7), in which polypeptide unfolding and cluster degra- dation occur simultaneously. At pH 10, however, the kinetic process monitored by visible absorption did not match that probed by far-UV CD (discussed below). Next, GdnHCl titrations at 20 °C were performed at various pH values, and FdTt unfolding was probed at different incubation times (from 2 to 48 h) using the three spectroscopic methods. The GdnHCl-induced unfolding process is irreversible at all pH values, probably because the clusters decompose on unfolding (in accord with complete visible-absorption disappearance, Fig. 2A). It is also possible that cysteine oxidation takes place in the unfolded state, hampering refolding. Irreversible unfolding has been reported for other ferredoxins [17–20]. Because of the irreversibility of the reaction, no thermodynamic data, such Fig. 2. Visible absorption (A), tryptophan emission (B), and far-UV CD (C) of native FdTt (solid line, 0 M GdnHCl,pH 7,20°C) and denatured FdTt (dotted line, 7.9 M GdnHCl 48 h incubation, pH 7, 20 °C). 4738 S. Griffin et al.(Eur. J. Biochem. 270) Ó FEBS 2003 as DG U (H 2 O), could be obtained for FdTt; instead, we report unfolding-transition midpoints as a function of pH and incubation time at 20 °C (summarized in Table 1). The transition midpoints shift to lower GdnHCl concentration as the incubation time is increased as expected for an irreversible reaction (Table 1). We observe single unfolding transitions with all spectro- scopic probes under all conditions except at high pH (see below). The different probing methods, reporting on different properties of FdTt, gave similar transition mid- points for FdTt samples incubated for the same amount of time and at the same pH condition (Table 1), supporting the suggestion that FdTt unfolding is a two-state process with cluster degradation occurring simultaneously with polypep- tide unfolding. For example, the midpoint of the unfolding transition appeared at  6.5 M GdnHCl after 48 h of incubation at pH 7 (20 °C) as monitored by all three probes. At pH values lower than 7, the apparent stability of FdTt decreased significantly (Fig. 4); for example, at pH 2.5, the midpoint of transition was 1.5 M GdnHCl (48 h incubation, 20 °C). Transition midpoints for FdTt samples with and without 500 m M NaCl were identical (20 °C, pH 7), indicating that protein stability is not affected by the presence of NaCl (data not shown). The effect of protein concentration was investigated in a separate experiment by comparing unfold- ing transitions for 10 l M and 80 l M FdTt at pH 2.5. The transition midpoints, monitored by visible absorption, was not significantly different for the two different protein concentrations. In this experiment (pH 2.5, 20 °C), the GdnHCl-induced midpoints were 1.7 and 1.6 M GdnHCl after 2 h of incubation and 1.1 and 1.0 M GdnHCl after 24 h of incubation for 80 and 10 l M FdTt, respectively. Thermally induced FdTt unfolding In buffer at pH 7, FdTt does not unfold below 100 °C. Therefore, thermally induced unfolding experiments were conducted in the presence of different concentrations of GdnHCl (not high enough to unfold FdTt at 20 °C). Like GdnHCl-induced unfolding at 20 °C, thermal unfolding of FdTt occurred in a single transition which was irreversible. In most thermal experiments, visible absorption was used as detection probe, although some experiments were also probed by far-UV CD and fluorescence. At each condition, identical T m values were observed with the different detection probes. For each scan rate studied (0.5, 0.25 and 0.13 °CÆmin )1 ), thermal midpoints (T m ) obtained at Fig. 3. FdTt unfolding kinetics (20 °C), measured by visible absorption at 408 nm, on addition of 7.9 M GdnHCl at pH 7 (solid line) and pH 2.5 (dashed line). Inset: far-UV CD detection of the same process (solid line,pH7;dashedline,pH2.5). Table 1. GdnHCl-induced unfolding midpoints (M) for FdTt at four pH values probed by visible absorption (Abs), tryptophan emission (FL), and far-UV CD (CD) after different incubation times (20 °C). The values have an error of ± 0.2 M . *, Intermediate with new absorption bands forms on unfolding; therefore, unfolding midpoints are not reliable by this technique. pH Time (h) Unfolding midpoints (M) Abs FL CD 2.5 2 2.2 2.3 2.2 8 2.0 2.2 2.2 24 1.6 2.0 1.7 48 1.5 1.5 1.5 4 2 4.7 4.7 4.5 8 4.2 4.4 3.9 24 3.8 4.0 3.8 48 3.5 3.7 3.5 7 2 No unfolding No unfolding No unfolding 8 7.1 7.3 7.4 24 6.7 7.0 6.8 48 6.4 6.5 6.6 10 2 * 6.2 6.2 8 * 5.6 5.8 24 * 5.6 5.6 48 * 5.5 5.4 Fig. 4. GdnHCl concentrations at which the midpoint of unfolding transitions occur as a function of pH. Incubation times 2 h (d), 8 h (j), 24 h (r), and 48 h (m). Ó FEBS 2003 Stability of T. thermophilus seven-iron ferredoxin (Eur. J. Biochem. 270) 4739 different concentrations of GdnHCl were extrapolated to give a T m value for each scan rate at 0 M GdnHCl. Next, these T m values at 0 M GdnHCl for different scan rates were extrapolated to (1/scan rate) ¼ 0, to give a T m correspond- ing to infinite scan rate conditions. The method of extrapolation to infinite scan rate has been used before to eliminate irreversible time-dependent steps in protein- unfolding reactions [21]. The thermal midpoints for FdTt unfolding (at infinite scan rate) are 69 °C (pH 2.5), 91 °C (pH 4), 114 °C (pH 7), and 90 °C (pH 10.5) (Fig. 5). Thus, optimum stability of FdTt to heat also occurs around neutral pH, and the thermal stability is, like the resistance to chemical denaturation, dramatically reduced at lower pH. Formation of linear clusters at high pH On GdnHCl-induced unfolding of FdTt at high pH (20 °C), new visible absorption bands at 520 nm and 610 nm appeared transiently before complete disappearance of the visible absorption occurred (Fig. 6A). The new peaks formed with rate constants that depended on the concentration of GdnHCl (pH 10, 20 °C). In 7.9 M GdnHCl, the new bands formed rapidly and became more intense (maximum intensity reached within 25 min) than at 6 M GdnHCl where the formation was slower (maximum intensity reached within 80 min). Identical absorption bands at 520 nm and 610 nm have been observed transi- ently in some other ferredoxins and in beef aconitase under various conditions that perturb the protein structure [19,20,22]. In those cases, it was concluded from EPR and Mo ¨ ssbauer studies and comparison with small model compounds [23] that the new absorption features resulted from linear three-iron clusters bound to the unfolded, or partially unfolded, polypeptide (Fig. 6A, inset). The formation of the new absorption bands for FdTt correlated with the disappearance of the far-UV CD signal, implying Fig. 5. (A) T m vs. concentration of GdnHCl at pH 7 for three different scan rates [(j)0.5°CÆmin -1 ;(d)0.25°CÆmin -1 ;(m)0.13°CÆmin -1 )] and (B) T m (at infinite scan rate) as a function of pH. Fig. 6. Formation of linear species at high pH. (A)Visible absorption of FdTt on addition of 7.9 M GdnHCl (pH 10.5) after 1 min (solid line), 15 min (dotted line), and 24 h (dotted-dashed line) incubation (inset: schematic drawing of a linear three-iron cluster). (B) EPR spectra (110 K, 10 mW, 9.3 GHz) of folded FdTt in pH 10.5 (thin line) and FdTt at pH 10.5 incubated for 15 min in 6.5 M GdnHCl (thick line; corresponding to dotted line in A). 4740 S. Griffin et al.(Eur. J. Biochem. 270) Ó FEBS 2003 that polypeptide unfolding triggered linear-cluster forma- tion. Support for the idea that the new absorption bands also correspond to linear three-iron clusters in FdTt comes from EPR experiments (Fig. 6B). At pH 10.5, FdTt exhibits a typical [3Fe)4S] cluster resonance at g ¼ 2.02 and a minor contribution at g ¼ 4.3 from adventitious iron in solution. On incubation in 6.5 M GdnHCl for 15 min (pH 10.5, 20 °C; to reach maximum intensity at 610 nm), the signal from the [3Fe)4S] center decreases with the concomitant 15-fold increase in the g ¼ 4.3 resonance and a small peak at g ¼ 9.5 (features charac- teristic of an S ¼ 5/2 system in a rhombic environment). The S ¼ 5/2 signal is compatible with the presence of a linear three-iron cluster and very similar to that reported for purple aconitase, another seven-iron ferredoxin, and model compounds [19,22,23]. The linear [Fe–S] cluster remained in the unfolded protein for several hours (5–10 h; 20 °C, pH 10.5, 6.5–7.0 M GdnHCl) before complete loss of visible absorption occurred. Discussion Understanding and probing protein stability at high temperatures and extreme conditions is relevant for a variety of biochemical and biotechnological applications. Intriguingly, the stability of a protein can be increased by the optimization of a few interactions without large structural modifications. To further understand the mechanisms of increased stability in bacterial ferredoxins, we characterized chemical and thermal denaturation of the seven-iron T. thermophilus ferredoxin (FdTt) in solution. This protein was recently crystallized [16], but in vitro solution studies have been lacking. The data presented here thus constitute the groundwork for future experiments in which strategic FdTt mutants, designed on the basis of the crystal structure, can be directly compared with the biophysical behavior of the wild-type form. Our solution study shows that wild-type FdTt is extremely stable to heat and chemical denaturants. An explanation for this behavior, in terms of the sum of many minor effects, is found on analyzing the crystal structure. FdTt shares 64% sequence identity with the mesophilic ferredoxin from Azotobacter vinelandii (FdAv), which is a protein with the same overall structure as FdTt but significantly less stable in solution [16]. Like other mesophilic ferredoxins, FdAv has a stretch of 29 residues at the C-terminus, which is absent from FdTt. This stretch of residues protects the [3Fe)4S] cluster in FdAv from solvent. Hence, the [3Fe)4S] cluster is more accessible to solvent in FdTt, and shielding of this cluster from solvent cannot be vital for FdTt function. Because FdTt has a shorter C-terminus than FdAv, it has less accessible solvent surfacearea,whichmayaidinresistancetoperturbation [11,12]. On comparing the crystal structures, polar residues at the surface of FdTt replace topologically equivalent negatively charged residues in FdAv. Moreover, an a-helix in FdTt, replacing a 3 10 -helix in FdAv, is stabilized with alanine residues [16], and the (bab) 2 core of FdTt is stabilized by additional hydrogen bonds between side chains and the main chain, as compared with FdAv. Also, FdTt has more glycine residues than FdAv, which may minimize conformational strain in the folded state [16]. FdAv uses a cluster of glutamic and aspartic acid residues to electrostatically interact with its physiological electron- transfer partner. In FdTt, this region of the protein’s surface is less charged. This difference in electrostatics is thought to increase FdTt stability by reducing unfavorable repulsions between negatively charged residues. The cor- responding reduced electrostatic attraction between FdTt and its electron-transfer partners may be compensated for by faster protein diffusion rates at the higher temperatures [16]. In addition to our biophysical study of FdTt presented here, five other thermostable seven-iron ferredoxins have been studied in vitro with respect to thermostability: from the thermostable bacteria Bacillus schlegelii [24] and Bacillus acidocaldarius [25] and the thermostable archaea Acidianus ambivalens [13,20] and Sulfolobus sp. strain 7 [26]. Three of these proteins (and FdTt) are stable above the boiling point of water at pH 7. Ferredoxin from B. schlegelii begins to unfold at 90 °C [24], ferredoxin from B. acidocaldarius is completely denatured at 88 °C [25,27], ferredoxin from A. ambivalens (FdA; species with zinc ion) has a thermal midpoint of 122 °C(pH7)[13], and another ferredoxin from A. ambivalens (FdB; species without zinc) has a thermal midpoint of 108 °C (pH 6.5) [18]. The seven-iron ferredoxin from Sulfolobus sp. strain 7 has a thermal midpoint of 109 °C [28]. FdTt, with its 114 °C thermal midpoint at pH 7, is thus one of the most stable seven-iron ferredoxins (in fact, among proteins in general) investigated to date. Our GdnHCl-induced unfolding data at 20 °C for FdTt can only be compared withsimilarworkontheA. ambivalens ferredoxin (FdA; species with zinc). In the case of that protein, the GdnHCl-induced unfolding midpoints appear at 7.1 M (pH 7, 20 °C), 2.3 M (pH 2.5, 20 °C), and 6.3 M (pH 10, 20 °C) [13]. Thus both proteins have similar extreme resistance to chemical denaturants at pH 7 and higher. The dramatic reduction in apparent stability at low pH for both FdTt and A. ambivalens ferredoxin (using both chemical and thermal perturbation) implies that electro- static interactions contribute significantly to the proteins’ integrity at the higher pH values. This occurs because, at low pH values, salt bridges are easily broken due to protonation of aspartic and glutamic acid residues (which have pK a values around 4). On GdnHCl-induced polypeptide unfolding at high pH, we find that the clusters in FdTt transiently rearrange into intermediate species before complete cluster degradation occurs. Clusters with the same spectroscopic features as we found in FdTt at high pH have been shown in other studies to be linear [3Fe)4S] clusters still bound to the polypeptides (Fig. 6A, inset) [19]. Our spectroscopic data (absorption and EPR) support the suggestion that in FdTt also the cubic clusters rearrange into linear species on protein unfolding under alkaline conditions. The presence of a linear [3Fe)4S] cluster was first observed in the protein bovine heart aconitase, at high pH where the protein structure was perturbed [22]. Recently, the same linear cluster was discovered on in vitro unfolding of seven-iron ferredoxins from A. ambivalens and S. acidoc- aldarius [13,19,20]. The observation of a linear cluster in FdTt and other seven-iron ferredoxins, and the recent Ó FEBS 2003 Stability of T. thermophilus seven-iron ferredoxin (Eur. J. Biochem. 270) 4741 observations of this cluster in [2Fe)2S] ferredoxins from Aquifex aeolicus ([17] and unpublished data), suggest a more general relevance of this type of linear cluster in nature. In Table 2, we summarize the known systems and solution conditions in which linear three-iron clusters have been observed in vitro. No biological function for linear [3Fe)4S] clusters is yet known, although reconstituted, recombinant human cytosolic iron regulatory protein 1 has been found to contain such a cluster under physio- logical conditions [29]. We speculate that [Fe–S] cluster rearrangements induced by protein-conformational chan- ges may be used for regulatory purposes in vivo.The linear cluster may be a storage or transport form for iron and sulfide in the cells ready for use in resynthesis of cubic (functional) clusters. Summary Examination of the crystal structure of the seven-iron ferredoxin from T. thermophilus has suggested that it represents the minimal functional unit of this type of protein[16].Inagreement,wefindFdTttobeaverystable protein in solution in vitro: temperatures above boiling or high denaturant concentrations and long incubation times are necessary to perturb it. From our work in solution at different pH values, it is clear that electrostatic interactions play a significant role in governing the high stability of FdTt. As unfolding is very slow (hours), even in high concentrations of denaturant, there appears to be a kinetic barrier to FdTt unfolding. Slow unfolding kinetics may be a general mechanism governing high stability of thermostable proteins. On polypeptide unfolding at high pH, linear three-iron clusters form in FdTt. Recent discoveries of the transient appearance of such linear clusters in many different iron–sulfur proteins imply that they may be of biological relevance. Acknowledgements This work was supported by the National Institute of Health (GM5966301A2) (P.W S.), the Louisiana Board of Regents (C.L.H.), and the Newcomb College Fellows Program (Tulane University, New Orleans, Louisiana) (S.G.). We thank Ah-lim Tsai (University of Texas Medical School, Houston) and John S. Olson (Rice University, Houston) for help with EPR experiments. References 1. Bertini, I., Cowan, J.A., Luchinat, C., Natarajan, K. & Piccioli, M. (1997) Characterization of a partially unfolded high potential iron protein. Biochemistry 36, 9332–9339. 2. Goedken, E.R., Keck, J.L., Berger, J.M. & Marqusee, S. (2000) Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI. Protein Sci. 9, 1914–1921. 3. Leckner, J., Bonander, N., Wittung-Stafshede, P., Malm- strom, B.G. & Karlsson, B.G. (1997) The effect of the metal ion on the folding energetics of azurin: a comparison of the native, zinc and apoprotein. Biochim. Biophys. Acta 1342, 19–27. 4. Pozdnyakova, I., Guidry, J. & Wittung-Stafshede, P. (2000) Copper triggered beta-hairpin formation. Initiation site for azurin folding? J. Am. Chem. Soc. 122, 6337–6338. 5. Wittung-Stafshede, P., Hill, M.G., Gomez, E., Di Bilio, A., Karlsson, G., Leckner, J., Winkler, J.R., Gray, H.B. & Malmstrom, B.G. (1998) Reduction potentials of blue and purple copper proteins in their unfolded states: a closer look at rack-induced coordination. J. Biol. Inorg. Chem. 3, 367–370. 6. Robinson, C.R., Liu, Y., Thomson, J.A., Sturtevant, J.M. & Sligar, S.G. (1997) Energetics of heme binding to native and de- natured states of cytochrome b 562 . Biochemistry 36, 16141–16146. 7. Frazzon, J. & Dean, R.D. (2001) Feedback regulation of iron- sulfur cluster biosynthesis. Proc. Natl Acad. Sci. USA 98, 14751–14753. 8. Mortenson, L.E., Valentine, R.C. & Carnahan, J.E. (1962) An electron transport factor from Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 7, 448–452. 9. Tagawa, K. & Arnon, D.I. (1962) Ferredoxins as electron carriers in photosynthesis and in the biological production and con- sumption of hydrogen gas. Nature (London) 195, 537–543. 10. Meyer, J. (2001) Ferredoxins of the third kind. FEBS Lett. 509, 1–5. 11. Jaenicke, R. & Bohm, G. (1998) The stability of proteins in extreme environments. Curr. Opin. Struct. Biol. 8, 738–748. 12. Vogt, G. & Argos, P. (1997) Protein thermal stability: hydrogen bonds or internal packing? Fold. Des. 2, S40–S46. 13. Moczygemba, C., Guidry, J., Jones, K., Gomes, C., Teixeira, M. & Wittung-Stafshede, P. (2001) High stability of a ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens: involve- ment of electrostatic interactions and cofactors. Protein Sci. 10, 1539–1548. 14. Szilagyi, A. & Zavodszky, P. (2000) Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Struct. Fold. Des. 8, 493–504. Table 2. Proteins found to accommodate linear three-iron clusters in vitro and the corresponding solution conditions. Protein Origin Normal cluster(s) Conditions for linear cluster Ferredoxin[this work] T. thermophilus [3Fe)4S]/[4Fe)4S] High pH and [GdnHCl] Ferredoxin [19] S acidocaldarius [3Fe)4S]/[4Fe)4S] High pH and [GdnHCl] Ferredoxin [20] A. ambivalens [3Fe)4S]/[4Fe)4S] High pH and [GdnHCl] Aconitase [22] Bovine heart [4Fe)4S] High pH or [urea] Dihydroxy acid dehydratase [30] E. coli [4Fe)4S] Exposure to oxygen Ferredoxin [31] R. marinus [3Fe)4S] High [GdnHCl] Iron regulatory protein 1 [29] Human cytosol [3Fe)4S] Physiological Fd1, Fd4 and Fd5 [17] a A. aeolicus [2Fe)2S] High pH and [GdnHCl] Ferredoxin a Spinach [2Fe)2S] High pH and [GdnHCl] Ferredoxin a C. pasteurianum [2Fe)2S] High pH and [GdnHCl] a Unpublished data. 4742 S. Griffin et al.(Eur. J. Biochem. 270) Ó FEBS 2003 15. Tabata, K., Kosuge, T., Nakahara, T. & Hoshino, T. (1993) Physical map of the extremely thermophilic bacterium Thermus thermophilus HB27 chromosome. FEBS Lett. 331, 81–85. 16. Macedo-Ribeiro, S., Martins, B., Pereira, P.J.B., Buse, G., Huber, R. & Soulimane, T. (2001) New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus. J. Biol. Inorg. Chem. 6, 663–674. 17. Higgins, C., Meyer, J. & Wittung-Stafshede, P. (2002) Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacter- ium Aquifex aeolicus. Biochim. Biophys. Acta 1599, 82–89. 18. Janssen, S., Trinca ˜ o, J., Teixeira, M., Scha ¨ fer, G. & Anemu ¨ ller, S. (2001) Ferredoxins from the archaeon Acidanus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB. Biol. Chem. 382, 1501–1507. 19. Jones, K., Gomes, C., Huber, H., Teixeira, T. & Wittung- Stafshede, P. (2002) Formation of a linear [3Fe-4S] cluster in a seven-iron ferredoxin triggered by polypeptide unfolding. J. Biol. Inorg. Chem. 7, 357–362. 20. Wittung-Stafshede, P., Gomes, C. & Teixeira, M. (2000) Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens. J. Inorg. Biochem. 78, 35–41. 21. La-Rosa, C., Milardo, D., Grasso, D., Guzzi, R. & Sprotelli, L. (1995) Thermodynamics of the thermal unfolding of azurin. J. Phys. Chem. 99, 14864–14870. 22. Kennedy,M.C.,Kent,T.,Emptage,M.,Merkle,H.,Beinert,H.& Mu ¨ nck, E. (1984) Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase. J. Biol. Chem. 259, 14463– 14471. 23. Hagen, K., Watson, A. & Holm, R.H. (1983) Synthetic routes to Fe 2 S 2 ,Fe 3 S 4 ,Fe 4 S 4 ,andFe 6 S 9 clusters from the common precursor [Fe (SC 2 H 5 ) 4 ] 2– : structures and properties of [Fe 3 S 4 (SR) 4 ] 3– and [Fe 6 S 9 (SC 2 H 5 ) 2 ] 4– , examples of the newest types of Fe-S-SR clusters. J. Am. Chem. Soc. 105, 3905–3915. 24. Aono, S., Bentrop, D., Bertini, I., Donaire, A., Luchinat, C., Niikura, Y. & Rosato, A. (1998) Solution structure of oxidized Fe 7 S 8 ferredoxin from thermophilic bacterium Bacillus schlegelii by H 1 NMR spectroscopy. Biochemistry 37, 9812–9826. 25. D’Auria, S., Rossi, M., Herman, P. & Lakowicz, J. (2000) Pyru- vate kinase from the Thermophilic eubacterium Bacillus acid- ocaldarius as probe to monitor the sodium concentrations in the blood. Biophys. Chem. 84, 167–176. 26. Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka,N.&Oshima,T.(1997)Thecrystalstructureofzinc- containing ferredoxin from the thermoacidophilic archaeon Sul- folobus sp. strain 7. Biochemistry 36, 1505–1513. 27. Schlatter, D., Waldvogel, S., Zu ¨ lli, F., Suter, F. & Portmann. & Zuber, H. (1985) Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius. Biol. Chem. Hoppe-Seyler 366, 223–231. 28. Kojoh, K., Matsuzawa, H. & Wakagi, T. (1999) Zinc and an N-terminal extra stretch of the ferredoxin from a thermo- acidophilic archaeon stabilize the molecule at high temperature. Eur. J. Biochem. 264, 85–91. 29. Gailer, J., George, G., Pickering, I., Prince, R., Kohlhepp, P., Zhang, D., Walker, A. & Winzerling, J. (2001) Human cytosolic iron regulatory protein 1 contains a linear iron-sulfur cluster. J. Am. Chem. Soc. 123, 10121–10122. 30. Flint, D., Emptage, M., Finnegan, M., Fu, W. & Johnson, M. (1993) The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase. J. Biol. Chem. 268, 14732–14742. 31. Pereira, M., Jones, K., Campos, M., Melo, A., Saraiva, L., Louro, R., Wittung-Stafshede, P. & Teixeira, M. (2002) A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus. Biochim. Biophys. Acta 1601,1–8. Ó FEBS 2003 Stability of T. thermophilus seven-iron ferredoxin (Eur. J. Biochem. 270) 4743 . High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin Linear clusters form at high pH on polypeptide unfolding Susanne. reduced at lower pH. Formation of linear clusters at high pH On GdnHCl-induced unfolding of FdTt at high pH (20 °C), new visible absorption bands at 520 nm and