Redox-linked ionization of sulredoxin, an archaeal Rieske-type [2Fe-2S] protein from Sulfolobus sp. strain 7.

“Sulredoxin” of Sulfolobus sp. strain 7 is an archaeal soluble Rieske-type [2Fe-2S] protein and was initially characterized by several spectroscopic techniques (Iwasaki, T., Isogai, T., Iizuka, T., and Oshima, T. (1995) J. Bacteriol. 177, 2576-2582). It appears to have tightly linked ionization affecting the redox properties of the protein, which is characteristic of the Rieske FeS proteins found as part of the respiratory chain. Sulredoxin had an Em(low pH) value of +188 ± 9 mV, and the slope of pH dependence of the midpoint redox potential indicated two ionization equilibria in the oxidized form with pKa(ox1) of 6.23 ± 0.22 and pKa(ox2) of 8.57 ± 0.20. The absorption, CD, and resonance Raman spectra of oxidized sulredoxin are consistent with the proposed St2FeSb2Fe[N(His)]t2 core structure, and deprotonation of one of the two putative coordinated histidine imidazoles, having the pKa(ox2) of 8.57 ± 0.20, causes a decrease in the midpoint redox potential, the change in the optical and CD spectra, and the appearance of a new Raman transition at 278 cm−1, without major structural rearrangement of the [2Fe-2S] cluster as well as the overall protein conformation. The redox-linked ionization of sulredoxin is also contributed by local changes involving another ionizable group having the pKa(ox1) of 6.23 ± 0.22, which is probably attributed to a certain positively charged amino acid residue that may not be a ligand by itself but located very close to the cluster. We suggest that sulredoxin provides a new tractable model of the membrane-bound homologue of the respiratory chain, the Rieske FeS proteins of the cytochrome bc1-b6f complexes.

The respiratory Rieske iron-sulfur (FeS) protein is an intrinsic constituent of cytochrome bc 1 -b 6 f complexes from mitochondria, chloroplasts, and certain bacteria found as part of the respiratory chain (1)(2)(3)(4). Although the Rieske-type FeS cluster consists of two Fe and two S 2Ϫ , it has a high midpoint redox potential and shows characteristic optical and EPR spectra that are distinctively different from those of the conventional plant-type ferredoxins in which the [2Fe-2S] cluster is bound to four sulfide ligands contributed by four cysteine residues and two bridging sulfide ions. The [2Fe-2S] clusters of several ferre-doxins involved in the bacterial dioxygenase systems also have spectral properties analogous to those of the respiratory Rieske FeS proteins (2). These spectral properties have been interpreted by the asymmetric ligand environments around the [2Fe-2S] cluster, such that one of its iron atoms is coordinated to the protein by two sulfide ligands contributed by two cysteine residues, while the other is coordinated by two nitrogens contributed by two putative histidine residues (4 -12).
The midpoint redox potentials of the mitochondrial and Rhodobacter sphaeroides Rieske FeS centers (ϩ280 and ϩ285 mV at pH 7.0, respectively) are independent of pH between 6 and 8 and decrease ϳ60 mV/pH above pH 8 (13). A similar redox-linked ionization effect has been reported for Thermus thermophilus Rieske FeS protein (ϩ140 mV at pH 7.0) (5,14) and a Rieske FeS cluster in Bacillus sp. PS3 (ϩ165 mV at pH 7.0) (15), both of which had the pK a(ox) of protonic equilibrium of ϳ8. The midpoint redox potential of the Rieske FeS center in a green sulfur bacterium Chlorobium limicola (ϩ160 mV at pH 7.0) decreases ϳ60 mV/pH from pH 6.8 to 8.4 (16), implying a significantly lower pK a in this particular case. On the other hand, the midpoint redox potentials of the Rieske-type [2Fe-2S] proteins involved in the bacterial dioxygenase systems are invariant, at least up to pH 10 (2,4,14). Thus, the respiratory Rieske FeS proteins involved in the cytochrome bc 1 -b 6 f complexes of the aerobic respiratory and photosynthetic systems have an "redox-linked ionization," usually with the pK a(ox) of the protonic equilibrium of ϳ8.
Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius strain 7) grows optimally at 80°C and at pH 2.5-3 and acquires biological energy by aerobic respiration rather than by simple fermentation, at least under the chemoheterotrophic growth conditions (17)(18)(19)(20)(21)(22)(23)(24). Since the archaeon contains only a-and b-type cytochromes but no c-types (17,22,23), it was unexpected that two different species of the thermoacidophilic archaea Sulfolobus contained the Rieske-type [2Fe-2S] centers (21,22,(25)(26)(27). In the case of Sulfolobus sp. strain 7, at least two different Rieske-type FeS centers have been detected in the membranes; one exhibiting the g y ϭ 1.89 EPR signal is a constituent of the archaeal respiratory terminal oxidase supercomplex, while the other exhibiting the g y ϭ 1.91 signal is of unknown function (21,22). The latter center is only loosely attached to the membrane and is readily removed by washing the membrane in the presence of cholate, 1 as in the cases of the cognate weakly associated membrane proteins such as NADH dehydrogenase (28) and V 1 -ATPase (18,29). In addition to these membrane-bound FeS centers, the archaeal soluble fraction exhibits another g y ϭ 1.91 EPR signal, which is attributed to a soluble purple FeS protein, tentatively called "sulredoxin." The initial characterization of sulredoxin by the absorption, CD, and EPR spectroscopic techniques, in conjunction with chemical analysis, suggested the presence of a single Riesketype [2Fe-2S] cluster in sulredoxin with an average g-factor of 1.90 (21). The size of sulredoxin determined by mass spectroscopy (12,155 Da (21)) is similar to those of the [2Fe-2S] proteins involved in the bacterial dioxygenase systems (12 kDa (2)) but much smaller than that of a membrane-bound respiratory Rieske FeS protein of S. acidocaldarius strain DSM 639 (32 kDa (27)).
Although the physiological function of sulredoxin remains unknown, as it did not function as an electron acceptor of the cognate NADH dehydrogenase (28), or the cognate ferredoxindependent enzymes such as 2-oxoacid:ferredoxin oxidoreductase (30) and NADPH:ferredoxin oxidoreductase (21), 1 it can be reproducibly purified in high purity and in the water-soluble and very stable form without detergents, thus being suitable for further physicochemical studies. In addition, because sulredoxin represents the only example of a soluble Rieske-type [2Fe-2S] protein so far purified from an archaeal species, it is of particular interest to investigate its redox property and to compare it with those of the mitochondrial and bacterial Rieske-type FeS proteins. In this paper, we report the potentiometric and the absorption, CD, and low temperature resonance Raman spectral properties of sulredoxin and discuss the nature of the redox-linked ionization of the archaeal Riesketype [2Fe-2S] protein. We suggest that sulredoxin provides a new tractable model of the membrane-bound Rieske FeS protein found as part of the respiratory chain.

EXPERIMENTAL PROCEDURES
Materials-Sulredoxin was routinely purified from the soluble fraction of Sulfolobus sp. strain 7 (an isolate from Beppu Hot Springs, Japan, originally named S. acidocaldarius strain 7) as described previously (21). Water was purified by the Milli-Q purification system (Millipore Corp.). Other chemicals mentioned in this study were of analytical grade.
Analytical Methods-Absorption spectra were recorded with a Shimadzu MP-2000 spectrophotometer or a Beckman DU-7400 spectrophotometer equipped with a thermoelectric cell holder. The visible near-UV and the far-UV CD spectra were recorded at room temperature on a Jasco J-720C spectropolarimeter connected to an NEC personal computer, in 0.5-or 0.1-cm cells, respectively.
The optical potentiometric titration of purified sulredoxin was performed at room temperature in a Thunberg-type cell essentially as described by Kuila and Fee (14) under continuous flow of argon gas and stirring, except in the presence of 1-2 M each of methyl viologen, anthraquinone-␤-sulfonic acid, phenazine methosulfate, duroquinone, 1,2-naphthoquinone, and vitamin K 3 as redox mediators. Ambient redox potentials (E h ) were monitored with a Pt-Ag/AgCl electrode (Beckman Instruments), and desired potentials were attained by adding a small volume of potassium ferricyanide or sodium dithionite solution. The obtained absorption spectra were recorded with a Shimadzu MP-2000 spectrophotometer. To minimize the effects of mediators, absorbance pairs at 490 and 550 nm were used for corrections of the base-line drift; the absorbance at 510 nm of each spectrum (see Fig. 2 in Ref. 21) was then analyzed for the calculation of the midpoint redox potentials from the titration curves using the KaleidaGraph software package (Abelbeck Software). The pH values of the protein solutions with the mediators were controlled at the beginning and at the end of each redox titration with small volumes of diluted acetate, HCl, or NaOH.
The optical and CD titrations were carried out with unbuffered sulredoxin sample (dissolved in Milli-Q water), whose pH values were carefully controlled at the beginning and at the end of each titration with small volumes of diluted acetate or NaOH, according to the guideline by Kuila and Fee (14). The protein concentrations of the sample used for the titration experiments were in a range of 0.12-0.47 mg/ml in distilled water.
The low temperature resonance Raman spectra were recorded at 77 K using 488.0 nm Ar ϩ laser excitation (500 milliwatts) essentially as described by Imai et al. (31). The highly concentrated sample was immersed into a liquid nitrogen reservoir, and the scattered light near 45°to the incident beam was collected. The spectral slit width was 4 cm Ϫ1 and a multiscan averaging technique was employed.
Purified sulredoxin was measured by the bicinchoninic acid assay (Pierce) with bovine serum albumin as a standard, and the results were divided by 2.8 for calibration (21).

RESULTS AND DISCUSSION
Potentiometric Titration of Sulredoxin- Fig. 1 shows the results of reductive potentiometric titration of purified sulredoxin performed over the pH range 5.2-9.8. All individual potentiometric titration curves could be fitted with simple n ϭ 1 Nernst equations (e.g. Fig. 1, inset). With increasing pH, the midpoint redox potential (E m ) decreases, and the slope around pH 7-8 is approximately 60 mV/pH, indicating a single deprotonation of oxidized sulredoxin under the conditions. At higher pH, however, the slope of the curve became much steeper, indicating the presence of a second pK a(ox) . On the other hand, the slope of the curve does not level off up to pH 10, indicating that the redox-linked deprotonation of the reduced protein does not occur. Thus, assuming purified sulredoxin shows a pH dependence of redox potential defined by two pK a(ox) in the oxidized state (cf. Ref. 32), the behavior of its titration curve is theoretically described using the methods of Clark (33).
While the data show an undesirable amount of scatter (of unknown origin) at low pH, the solid trace in Fig. 1 is a least squares fit to this equation, giving the E m(low pH) of ϩ188 Ϯ 9 mV, the pK a(ox1) of 6.23 Ϯ 0.22, and the pK a(ox2) of 8.57 Ϯ 0.20 (r ϭ 0.996). For comparison, a least squares fit to the data assuming only one pK a(ox) value clearly does not describe the pH-dependent redox behavior of sulredoxin correctly (the E m(low pH) of ϩ207 Ϯ 40 mV, and the pK a(ox) of 5.44 Ϯ 0.74 (r ϭ 0.964); dotted trace in Fig. 1). These data suggest that the midpoint redox potential of sulredoxin (E m(low pH) , ϩ188 mV) is influenced by two ionization equilibria with pK a(ox1) of 6.23 Ϯ 0.22 and pK a(ox2) of 8.57 Ϯ 0.20.
Optical Spectra of Sulredoxin- Fig. 2 shows the effect of pH on the visible absorption spectrum of oxidized sulredoxin. The optical titration showed a single pK a value of 8.4 by fitting the pH dependence of the amplitude of the 432 nm absorbance band, indicative of a single deprotonation. This value is very similar to that obtained from the pH dependence of the mid- The pH dependence of the midpoint redox potential of the Sulfolobus sulredoxin described by a least squares fit with the following parameters (solid trace) is shown: E m (low pH), ϩ188 mV; pK a(ox1) , 6.23; pK a(ox2) , 8.57; r ϭ 0.996. The slope at pH 10 is ϳϪ120 mV/pH. For comparison, a fit with a single pK a(ox) value (E m (low pH), ϩ207 mV; pK a(ox) , 5.44; r ϭ 0.964) is also shown (dotted trace). Inset, potentiometric titration of purified sulredoxin (ϳ0.5 mg/ml) at pH 6.54, which yielded the midpoint redox potential, E m ϭ ϩ155 mV (n ϭ 1). The ambient redox potential was changed by adding a small amount of dithionite solution. For other conditions, see "Experimental Procedures." point redox potential (Fig. 1). Similar spectral changes have been reported for T. thermophilus Rieske FeS protein (10,14) and the mitochondrial Rieske fragment (32). On the other hand, no significant pH-dependent spectral changes of the amplitude of the 432 nm absorbance band could be detected over pH 4.9 -7.0 (Fig. 2, inset). These data suggest that the visible absorption spectrum of oxidized sulredoxin is affected by the pK a(ox2) (obtained from the pH dependence of the E m values; Fig. 1) but not by the pK a(ox1) .
The optical titration of dithionite-reduced sulredoxin was also carried out in the range of pH 5.5-9.2, and the visible spectra obtained in the region were found superimposable (data not shown). In conjunction with the pH dependence of the potentiometric properties of sulredoxin (Fig. 1), this suggests that the reduced form of the [2Fe-2S] cluster of sulredoxin is always protonated.
Circular Dichroism Spectra of Sulredoxin-Since visible CD spectrum is very sensitive to changes in the orientation (conformation) and the dipole strength (bonding) of metal chromophores in metalloproteins (34), the pH-dependent CD spectral change of oxidized sulredoxin was further investigated to clarify whether the pK a(ox1) indeed does not affect the conformation and the structure of the [2Fe-2S] cluster. Fig. 3 shows the pH dependences of the visible and near-UV CD spectra of oxidized sulredoxin. The CD spectrum is distinctively different between acid-neutral pH and alkaline pH. At pH below 7, the electronic transitions resolved in the CD spectrum showed no drastic dichroic spectral change (Fig. 3, inset). Thus, although there is evidence that the electron density in the [2Fe-2S] cluster of oxidized sulredoxin is changed at pH below 7 (Fig. 1), the orientation and the dipole strength of the [2Fe-2S] cluster are probably not considerably affected by the pK a of the first transition, pK a(ox1) of 6.23 Ϯ 0.22 (Fig. 3). This ionizable group apparently corresponds to that with the pK a(ox) of 9.2 reported for the mitochondrial Rieske fragment (32). No significant change was observed in the near-UV CD spectrum of oxidized sulredoxin at pH below 7, excluding a possibility of the involvement of any aromatic residue in the first transition (Fig. 3).
On the other hand, inspection of the CD spectral changes at pH above 7 suggested two types of the pH-transition dependences. Upon deprotonation, the relative intensities of the optical activities of the 290, 312, and 458 nm positive bands decreased while those of the 422 and 486 nm positive bands increased. The pH dependences of the former dichroic spectral changes were fitted to a single pK a value of 8.4, and the latter changes were fitted to a single pK a value of 8.0 (Fig. 3, inset). Although it is not possible to assign the nature of each electronic transition in the CD spectrum, the marked changes in the optical activities of oxidized sulredoxin upon deprotonation may reflect either strengthening or weakening of certain bond strengths that are associated with the [2Fe-2S] cluster and/or the amino acid residues surrounding it. The average of the apparent pK a values obtained from the pH-transition dependences of the CD spectrum of oxidized sulredoxin, 8.22 Ϯ 0.23, is similar to that obtained from the visible absorption titration (Fig. 2) and is attributed to the pK a(ox) of the second transition detected in the pH dependence of the midpoint redox potential, pK a(ox2) of 8.57 Ϯ 0.20 (Fig. 1). This ionizable group apparently corresponds to that with the pK a(ox) of 7.6 of the mitochondrial Rieske fragment (32) and that with the pK a(ox) of ϳ8 of T. thermophilus Rieske FeS protein (14) and is further investigated by the low temperature resonance Raman spectroscopy (see below).
Low Temperature Resonance Raman Spectra of Sulredoxin-The resonance Raman spectroscopy has been extensively utilized to investigate the characteristics of [2Fe-2S] clusters in various FeS proteins (35)(36)(37)(38)(39) and has been applied to two Rieske-type [2Fe-2S] proteins, viz. T. thermophilus Rieske FeS protein and Pseudomonas cepacia phthalate dioxygenase (8,10). We have applied this technique for the first time to an archaeal Rieske-type [2Fe-2S] protein in order to obtain structural information of the [2Fe-2S] cluster of oxidized sulredoxin at different pH values. Fig. 4 shows that the resonance Raman spectra of oxidized sulredoxin recorded at 77 K and at pH 5.5, 7.4, and 9.4. At least 9 -10 peaks could be detected in the 230 -450 cm Ϫ1 region, and the overall features in this region are remarkably similar to those of other Rieske FeS proteins (8,10). This clearly indicates the similarities of the [2Fe-2S] core structures among these proteins. The appearance of more peaks in Rieske FeS proteins than the case of spinach ferredoxin (seven well documented peaks in the latter protein (36,40)) is expected by considering the symmetry of [Fe 2 S b 2 ]S t 4 , where S b is bridging sulfide and S t is terminal mercaptide sulfur from cysteine, compared with that of the proposed S t 2 FeS b 2 Fe[N(His)] t 2 structure with C 2v symmetry (8,10). The Fe(III)-N stretches are expected to be in the 200 -300 cm Ϫ1 region, assuming histidine imidazole coordination to high spin Fe(III). Kuila et al. (10) suggested that a band near 270 cm Ϫ1 , consisting of components at 266 cm Ϫ1 and 274 cm Ϫ1 whose relative populations are pH-dependent, is attributed to Fe(III)-N(His) t stretching motions of T. thermophilus Rieske FeS protein.
The resonance Raman spectra of oxidized sulredoxin exhibit several significant changes upon raising the pH from 5.5 to 9.4. In the 230 -300 cm Ϫ1 region, there is a single band at 270 cm Ϫ1 at pH 5.5-7.4, and at higher pH values the 278 cm Ϫ1 band appears so that at pH 9.4 both are present in the spectrum. Thus, these two bands at 270 and 278 cm Ϫ1 can be primarily attributed to Fe(III)-N(His) t stretching motions of sulredoxin, and the appearance of the new 278 cm Ϫ1 band at alkaline pH may be due to the strengthening of the Fe-N bond upon deprotonation. An additional weak Raman band at 297 cm Ϫ1 , which is virtually pH-independent, might also be due to a Fe(III)-N(His) t stretch, but this remains to be clarified. Another change observed in the pH-dependent manner is disappearance of a weak peak at 424 cm Ϫ1 at pH above 7.4, which may be due to differing resonance enhancement of arising observed pH dependence of the absorption spectrum (cf. Ref. 10).
The resonance Raman spectra of oxidized sulredoxin also exhibit several peaks below 370 cm Ϫ1 that are analogous to the terminal Fe-S stretches observed in the 330 -370 cm Ϫ1 region in spinach ferredoxin (36,40). In addition, as in the cases reported for other Rieske FeS proteins (8), there are at least two bands in sulredoxin at ϳ390 and 403 cm Ϫ1 , in place of the single symmetric Fe 2 S 2 ring stretching mode near ϳ390 cm Ϫ1 in spinach ferredoxin. These peaks are probably attributed primarily to Fe-S vibrations and are virtually independent of pH in the range 5.5-9.4.
Thus, several significant changes could be detected in the resonance Raman spectra of sulredoxin upon raising pH above 7.4, including the appearance of the new Raman transition at 278 cm Ϫ1 that is primarily attributed to the strengthening of an Fe(III)-N(His) t bond upon deprotonation. A similar observation has been reported for T. thermophilus respiratory Rieske FeS protein but not for P. cepacia phthalate dioxygenase or spinach ferredoxin (8). This clearly demonstrates the structural similarity of a [2Fe-2S] cluster of sulredoxin to those of the mitochondrial and bacterial respiratory Rieske FeS proteins with the proposed S t 2 FeS b 2 Fe[N(His)] t 2 structure. The resonance Raman spectroscopic analysis also demonstrates that the redox-linked ionization of sulredoxin is not accompanied by a major structural rearrangement of the [2Fe-2S] core structure (Fig. 4). The absence of the pH-dependent major conformational alteration is supported by the far-UV CD spectrum of oxidized sulredoxin. It showed the troughs at 206 and 221 nm, suggesting the presence of ␣-helical secondary structure and random coil, and is virtually identical in the pH range of 4.9 -9.6 (data not shown).
Possible Nature of Two Ionizable Groups in Sulredoxin-On the basis of the potentiometric and spectral properties of sulredoxin presented here, it can be concluded that deprotonation of one of the histidine imidazoles (having the pK a(ox2) of 8.57 Ϯ 0.20) in the putative S t 2 FeS b 2 Fe[N(His)] t 2 cluster in sulredoxin causes a decrease in the midpoint redox potential (Fig. 1), the change in the optical and CD spectra (Figs. 2 and 3), and the appearance of the new Raman transition at 278 cm Ϫ1 (Fig. 4). Sulredoxin also contains another ionizable group with a pK a(ox1) of 6.23 Ϯ 0.22, which may not be a ligand by itself but may be located in the vicinity of the oxidized [2Fe-2S] cluster. Deprotonation of this positively charged group causes a decrease in the midpoint redox potential (Fig. 1) but no significant changes in the orientation and relative strength of transition dipoles resolved in the visible and near-UV CD spectrum (Fig. 3) or the vibration modes in the resonance Raman spectrum (Fig. 4).
Recent studies by Link et al. (32) suggest the presence of two ionizable groups with the pK a(ox) of 7.6 and 9.2, respectively, in the vicinity of the [2Fe-2S] cluster of the water-soluble fragment of the respiratory Rieske FeS protein from bovine heart mitochondrial cytochrome bc 1 complex. The first positively charged group of the mitochondrial Rieske fragment having the apparent pK a of 7.6 causes a decrease in the midpoint redox potential and a change in the optical spectrum upon deprotonation and is proposed to be one of two putative histidine ligands to the [2Fe-2S] cluster; the second ionizable group is more basic (an apparent pK a of 9.2) and causes a decrease in the midpoint redox potential but no change in the optical spectrum upon deprotonation. The presence of the corresponding ionizable groups in sulredoxin indicates that the environments of the [2Fe-2S] cluster of the mitochondrial and archaeal Rieske FeS proteins are probably similar, 2 although the apparent pK a of one of the coordinating histidine imidazoles of the 2 In this connection, it is intriguing that a membrane-bound Riesketype FeS protein from another thermoacidophilic archaeon S. acidocaldarius strain DSM 639 (25,27) also contains two ionizable groups having the apparent pK a(ox) of 6 and 8.5, respectively, both of which cause a decrease in the midpoint redox potential upon deprotonation (43), as in the case of sulredoxin reported here. Although the nature of these ionizable groups in this protein has not been characterized in details (43), we suggest that the [2Fe-2S] cluster microenvironments of the archaeal Rieske-type FeS proteins may be very similar.
FIG. 4. Resonance Raman spectra of oxidized sulredoxin at pH 5.5, 7.4, and 9.4. The spectra were recorded at 77 K with 488.0 nm excitation and 500 milliwatts power and are an average of 8 scans. The samples were prepared essentially as described previously by Imai et al. (31). The scan rate was 10 cm Ϫ1 /min with a spectral resolution of 4 cm Ϫ1 .