Negatively charged residues and hydrogen bonds tune the ligand histidine pKa values of Rieske iron-sulfur proteins

Rieske proteins carry a redox-active iron-sulfur cluster, which is bound by two histidine and two cysteine side chains. The reduction potential of Rieske proteins depends on pH. This pH dependence can be described by two pK(a) values, which have been assigned to the two iron-coordinating histidines. Rieske proteins are commonly grouped into two major classes: Rieske proteins from quinol-oxidizing cytochrome bc complexes, in which the ligand histidines titrate in the physiological pH range, and bacterial ferredoxin Rieske proteins, in which the ligand histidines are protonated at physiological pH. In the study presented here, we have calculated pK(a) values of the cluster ligand histidines using a combined density functional theory/continuum electrostatics approach. Experimental pK(a) values for a bc-type and a ferredoxin Rieske protein could be reproduced. We could identify functionally important differences between the two proteins: hydrogen bonds toward the cluster, which are present in bc-type Rieske proteins, and negatively charged residues, which are present in ferredoxin Rieske proteins. We removed these differences by mutating the proteins in our calculations. The Rieske centers in the mutated proteins have very similar pK(a) values. We thus conclude that the studied structural differences are the main reason for the different pH-titration behavior of the proteins. Interestingly, the shift caused by neutralizing the negative charges in ferredoxin Rieske proteins is larger than the shift caused by removing the hydrogen bonds toward the cluster in bc-type Rieske proteins.     

Roles of the disulfide bond and adjacent residues in determining the reduction potentials and stabilities of respiratory-type Rieske clusters

Rieske [2Fe-2S] clusters have reduction potentials which vary by over 500 mV, and which are pH dependent. In the cytochrome bc(1) complex, the high-potential and low-pK values of the cluster may be important in the mechanism of quinol oxidation. Hydrogen bonds, from both side-chain and mainchain groups, are crucial for these properties, but solvent accessibility and a disulfide bond (present in only high-potential Rieske proteins) have been suggested to be important determinants also. Previous studies have addressed the hydrogen bonds, disulfide bond, and a leucine residue which may restrict solvent access, by mutations in the cytochrome bc(1) complex. However, influences on the complex (disruption of quinol binding and displacement of the Rieske domain) are difficult to deconvolute from intrinsic effects on the Rieske cluster. Here, the effects of similar mutations on cluster potential, pK values, and stability are characterized comprehensively in the isolated Rieske domain of the bovine protein. Hydrogen bonds from Ser163 and Tyr165 are important in increasing the reduction potential and decreasing the pK values. The disulfide has a limited effect on the redox properties, but is crucial for cluster stability, particularly in the oxidized state. Mutations of Leu142 had little effect on cluster potential, pK values, or stability, in contrast to the significant effects which were observed in the complex. The sum of the effects of all the mutated residues accounts for most of the differences between high- and low-potential Rieske proteins.     

Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.     

The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR.

The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.     

Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.     

A comparative,two-dimensional <Superscript>14</Superscript>N ESEEM characterization of reduced [2Fe–2S] clusters in hyperthermophilic archaeal high- and low-potential Rieske-type proteins

Proteins of the Rieske and Rieske-type family contain a [2Fe–2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome bc-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. ¹⁴N ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8–10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775–004–0571–y.Abbreviations ARF archaeal low-potential Rieske-type ferredoxin from Sulfolobus solfataricus - E_m midpoint redox potential - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - ESEEM electron-spin echo envelope modulation - hfi hyperfine interaction - HYSCORE hyperfine sublevel correlation - N1_SDX/ARF coordinated N in SDX and ARF with smaller isotropic hyperfine constant - N2_SDX/ARF coordinated N in SDX and ARF with larger isotropic hyperfine constant - nqi nuclear quadrupole interaction - SDX archaeal high-potential Rieske protein (sulredoxin) from Sulfolobus tokodaii - dq double quantum - sq single quantum - 1D one-dimensional - 2D two-dimensional     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability

Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pK(a) shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that (13)C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.     

The Qo-site inhibitor DBMIB favours the proximal position of the chloroplast Rieske protein and induces a pK-shift of the redox-linked proton.

The interaction of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b6f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron-sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske protein's conformational substates, strongly favouring the proximal position close to heme bL. In addition to this conformational effect, DBMIB shifted the pK-value of the redox-linked proton involved in the iron-sulphur cluster's redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.     

The Small Subunit AroB of Arsenite Oxidase: LESSONS ON THE [2Fe-2S] RIESKE PROTEIN SUPERFAMILY

Here, we describe the characterization of the [2Fe-2S] clusters of arsenite oxidases from Rhizobium sp. NT-26 and Ralstonia sp. 22. Both reduced Rieske proteins feature EPR signals similar to their homologs from Rieske-cyt b complexes, with g values at 2.027, 1.88, and 1.77. Redox titrations in a range of pH values showed that both [2Fe-2S] centers have constant E_m values up to pH 8 at ∼+210 mV. Above this pH value, the E_m values of both centers are pH-dependent, similar to what is observed for the Rieske-cyt b complexes. The redox properties of these two proteins, together with the low E_m value (+160 mV) of the Alcaligenes faecalis arsenite oxidase Rieske (confirmed herein), are in line with the structural determinants observed in the primary sequences, which have previously been deduced from the study of Rieske-cyt b complexes. Since the published E_m value of the Chloroflexus aurantiacus Rieske (+100 mV) is in conflict with this sequence analysis, we re-analyzed membrane samples of this organism and obtain a new value (+200 mV). Arsenite oxidase activity was affected by quinols and quinol analogs, which is similar to what is found with the Rieske-cyt b complexes. Together, these results show that the Rieske protein of arsenite oxidase shares numerous properties with its counterpart in the Rieske-cyt b complex. However, two cysteine residues, strictly conserved in the Rieske-cyt b-Rieske and considered to be crucial for its function, are not conserved in the arsenite oxidase counterpart. We discuss the role of these residues.     

Counteracting osmolyte trimethylamine N-oxide destabilizes proteins at pH below its pKa. Measurements of thermodynamic parameters of proteins in the presence and absence of trimethylamine N-oxide

Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.     

A Novel Rieske Iron-Sulfur Protein from the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum: Sequencing of the Gene, Expression in E. coli and Characterization of the Protein

The crenarchaeon Pyrobaculum aerophilum is with an optimalgrowth temperature of 100 °C one of the most thermophilic organisms knownto possess an aerobic respiratory chain. The analysis of DNA sequences fromthe Pyrobaculum genome project lead to the identification of an openreading frame potentially coding for a Rieske iron-sulfur protein. Thecomplete gene (named parR) was cloned and sequenced. The deducedamino acid sequence displays unusual amino acid exchanges and a so farunknown sequence insertion. The N-terminus shows similarities to bacterialsignal sequences. Several forms of the gene were expressed in E.coli in order to verify the classification as a Rieske protein and tofacilitate biophysical studies. Soluble, thermo-stable proteins withcorrectly inserted iron-sulfur clusters were expressed from two versions ofthe gene. The 1–23 truncated holo-protein is redox active. Itdisplays the typical spectroscopic properties of a Rieske protein. The redoxpotential was determined to be +215 mV at pH 6.5 and is pH dependentabove pH 7.5 revealing the influence of two protonation equilibria with pKavalues of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parRprotein clusters together with the two other available archaeal Rieskesequences from Sulfolobus on a separate branch of the phylogenetictree apart from the proteins from thermophilic bacteria like Aquifexand Thermus.     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.     

A Comprehensive Phylogenetic Analysis of Rieske and Rieske-Type Iron-Sulfur Proteins

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.     

Two-dimensional gel resolution of polypeptides specific for autotrophic growth in Thiobacillus versutus

Seven polypeptides specific for autotrophic growth by oxidation of thiosulphate have been identified in proteins of Thiobacillus versutus analysed by two-dimensional gel electrophoresis. Their molecular weights ranged from 32 to 100 kD and PI values from pH 6 to pH 7. Their probable relationships to previously reported Thiobacillus proteins involved in autotrophy, in particular ribulose bis-phosphate carboxylase, are discussed. These polypeptides will serve as useful landmarks in genetic study and manipulation of sulphur oxidation in these organisms.     

Rieske Iron–Sulfur Proteins from Extremophilic Organisms

Proteins located on the outside of the membranes of organisms thriving under extreme conditions like high or low pH, or high salinity face special challenges maintaining their structural integrity. This review is focused on the Rieske iron-sulfur proteins from these organisms. Rieske proteins are essential subunits of the cytochrome bc-complexes, which are often of crucial importance for the energy metabolism of the cells. On the basis of the available data we propose strategies by which these proteins are able to stabilize their noncovalent bound cofactor and adapt to the function under extreme conditions.     

A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b6/f (cyt b6/f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b6/f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b6/f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane deltapH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the pK and/or the redox potential of the oxidised Rieske protein.     

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. I. Characterization of the cytochrome bc1-type complex of the acidophilic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans.

The redox components of the cytochrome bc1 complex from the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans were investigated by potentiometric and spectroscopic techniques. Optical redox titrations demonstrated the presence of two b-type hemes with differing redox midpoint potentials at pH 7.4 (-169 and + 20 mV for bL and bH, respectively). At pH 3.5, by contrast, both hemes appeared to titrate at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of the b hemes' alpha-bands, providing evidence for the binding of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near heme bH (located on the cytosolic side of the membrane) and of stigmatellin near heme bL (located on the periplasmic side of the membrane). The inhibitors stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1 or b6f complexes. The results obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for bc1 complexes, contains unique properties that are most probably related to the chemolithotrophicity and/or acidophilicity of its parent organism. A speculative model for reverse electron transfer through the T. ferrooxidans complex is proposed.