Influence of anxiety on temporomandibular disorders--an epidemiological survey with elders and adults in Southern Brazil.

The socio-economic and psychological factors and the use of and need for dental prostheses have been associated with prevalence and severity of temporomandibular disorders (TMD). The aim of this study was to evaluate the association of socio-economic and psychological factors, use of and need for dental prostheses with the prevalence and severity of signs and symptoms of temporomandibular disorders (TMD). A cross-sectional study was conducted in Luzerna/Brazil. All individuals aged 35-44 (adults) and 65-74 (elders) were invited to participate in this study. The measuring instrument included a questionnaire application and the clinical evaluation regarding signs and symptoms of TMD. Descriptive analyses, chi-squared test, unadjusted and adjusted Poisson regression were used for the statistic analysis. The level of statistical significance was given when P ≤ 0·05. In total, 568 individuals (338 adults and 230 elders) were included; among them, 43·5% had absent, 42·6% mild, 11·3% moderate and 2·6% severe TMD. The presence of higher TMD rates was found in females (P = 0·001), and the TMD occurrence increased with anxiety level (P = 0·001). Other socio-economic factors, such as colour (P = 0·115), family structure (P = 0·478), age (P = 0·143), social class (P = 0·935) and education (P = 0·678) showed no influence. In conclusion, the female gender and individuals with higher levels of anxiety had increased prevalence of signs and symptoms of TMD. Additionally, was not founded association between TMD and use of and need for dental prostheses.

Cross-cultural adaptation of the orthognathic quality of life questionnaire (OQLQ) in a Brazilian sample of patients with dentofacial deformities.

OBJECTIVES: The aim of this study is to translate into Portuguese-Brazilian language and adapt cross-culturally to the Brazilian population the Orthognathic Quality of Life Questionnaire (OQLQ). STUDY DESIGN: The cross-cultural adaptation process followed six stages which are; (I) initial translation, (II) synthesis of the translation, (III) back translation, (IV) expert committee and (V) test of the prefinal version. For validation process, the OQLQ results were compared with Oral Health Impact Profile Questionnaire (OHIP-49), with the generic SF-36 Quality of Life Questionnaire and a visual analogue scale. A convenience sample of 25 patients was selected in two Southern Brazilian states of Santa Catarina and Rio Grande do Sul. RESULTS: Internal consistency analysis of the OQLQ-Brazilian showed good correlation for items or domains and the test-retest reliability also presented excellent intra-class correlation coefficients. OQLQ-Brazilian exhibited a weak and negative correlation with SF-36, and good correlation with OHIP-49. CONCLUSION: The OQLQ-Brazilian preserved and reached equivalence with its original source and the findings also corroborate that there is good evidence for the construct validity. The demonstration of its reproducibility, reliability and validity makes this instrument an additional useful parameter for evaluation of the impact of dentofacial deformity over the quality of life for Brazilians.

Effect of repetition of Semont's manoeuvre on benign paroxysmal positional vertigo of posterior semicircular canal.

If Semont's liberating manoeuvre does not lead to relief of symptoms in benign paroxysmal positional vertigo of posterior semicircular canal after the first session, it can be repeated once again, in refractory cases, whilst symptomatic patients after second manoeuvre require rehabilitation therapy Repeating Semont's manoeuvre several times has proven to progressively increase the percentage of cured patients or it may convert posterior semicircular canal forms to typical incomplete or lateral semicircular canal forms, hence requiring other manoeuvres to achieve vertigo resolution. Aim of study was to assess the effect of liberating manoeuvres repeated up to 4 times and to establish possible passages from one canal to the other during manoeuvres as well as percentage of cases refractory to this therapy, who would then need rehabilitation. Benign paroxysmal positional vertigo was diagnosed in 448 cases of whom 344 (76.8%) of the posterior semicircular canal, 20 (0.45%) the incomplete form of the posterior semicircular canal, 20 (0.45%) subjective positional vertigo and 74 of the lateral semicircular canal (4.2%). Right side was affected in 58.4% of cases, left in 34.5%, and bilateral in 7.1%. All 344 patients underwent Semont's liberating manoeuvre (1st manoeuvre) with first control after 48 hours: if symptoms (typical, atypical nystagmus or paroxysmal vertigo evoked by Dix-Hallpike's manoeuvre) persisted, Semont's liberating manoeuvre was repeated (2nd manoeuvre). In presence of lateral semicircular canal benign paroxysmal positional vertigo conversion, Lempert's manoeuvre was performed instead. Second control was performed after 48 hours and in cases of persistent typical, atypical or lateral semicircular canal nystagmus 3rd manoeuvre was performed. After further 48 hours, third control was carried out: symptomatic patients with typical forms were submitted to 4th manoeuvre, while typical incomplete forms or forms of the lateral semicircular canal underwent Lempert's manoeuvre. In conclusion, symptoms disappeared after 1st manoeuvre in 61.6% of cases; further manoeuvres, carried out in view of possible changes in semeiology of vertigo, increased the percentage of cured patients to 82.5% after the 2nd, 90.7% after 3rd and 94.1% after the 4th. Repeated positioning manoeuvres in benign paroxysmal positional vertigo led to a progressive increase in percentage of cured vertigo, at the same time, allowing detection of those cases converted to multicanal pathology, hence offering the possibility to proceed with appropriate liberating manoeuvres.

[Combined genioglossus advancement (ACMG): inferior sagittal mandibular osteotomy with genioglossus advancement and stabilization with suture in patients with OSAS. Preliminary clinical results]. / Avanzamento combinato del muscolo genioglosso (ACMG): osteotomia infero-sagittale mandibolare con avanzamento muscolo genioglosso e sua stabilizzazione con sutura in pazienti con OSAS. Risultati clinici preliminari.

Surgical treatment of obstructive sleep apnea syndrome (OSAS) centres on the identification of the level of obstruction of the upper airway and the choice of the most suitable procedure to correct it. Shaping of the retrolingual hypopharyngeal space is among the most difficult to achieve because it stems from an alteration of the soft tissue of the tongue, from the hypopharynx, and is correlated to the contraction pattern of the genioglossus and the pharyngeal constrictors. We propose a surgical technique of combined genioglossus advancement (CGA) in cases of anteroposterior collapse of the retrolingual airway. Four patients affected by OSAS (RDI average = 22 events/hour), evaluated as type III obstruction in the Fujita classification, presenting the indications for surgical management of retrolingual hypopharingeal obstruction, underwent treatment. They were studied by means of a guided medical history, fiberopy endoscopy evaluation and Muller maneuver, cephalometry, endocrine tests, pneumological examinations and polysomnography. The technique proposed consists in the advancement of the genioglossus muscle by means of a bone screw on the mandibular symphysis, according to the method described by Powell, associated with the stabilization of the base of the tongue with a suspension suture, following the technique originally described by the Author and DeRowe, but without using the Repose kit. This technique makes it possible to access the retrolingual site of obstruction more effectively, more economically and with no increase in morbidity when compared with the individual techniques. In all of the patients, the only complaints regarded dysaesthesia in the area of the lower lip innerved by the mental nerve for 2-5 weeks and moderate odynophagia for 2-3 weeks; there were no haemorrhages or infections. Deglutition of fluids and solids was resumed on the 3rd post-operative day. Polysomnography after 6 months documented three positive results and one partial result, on the basis of Sher's criteria. In conclusion, the CGA technique calls for advancement of the genioglossus insertion tubercle and stabilization of the tongue to be carried out at the same time, without using the Repose kit. The CGA technique is minimally invasive and does not involve cutaneous incisions, making it a therapeutic strategy which may be inserted in a multilevel protocol excluding transcutaneous access. It is therefore proposed for type III or type IIb cases in the Fujita classification.

Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase.

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.

Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction.

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H(4)B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H(4)B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.

[A new mini-invasive technique for the treatment of sleep breathing disorders: preliminary results of a clinical experience]. / Nuova tecnica mini-invasiva per il trattamento dei disturbi respiratori del sonno (DRS). Risultati preliminari della nostra esperienza clinica.

The REPOSE system is a new, mini-invasive technique with which the base of the tongue is suspended to treat sleep breathing disorders (SBD) induced by hypertrophy of the base of the tongue. The surgical technique calls for the intra-oral insertion of a small titanium screw in the anterior portion of the mandible. Two polypropylene threads are attached to the screw and these are passed through the base of the tongue and then tied at the point where it is inserted in the floor of the mouth, thus effectively suspending the base of the tongue. 10 patients with SBD due to hypertrophy of the base of the tongue underwent this procedure. Only one major complication was found: an infection requiring sectioning of the suspension thread. For an average 7 to 30 days all patients showed signs of odinophagia, bilateral otalgia, dysphagia and dislalia. In all patients snoring either disappeared altogether or was significantly reduced. Statistical analysis of the pre- and post-operative polysonnograph data showed a significant reduction in the apnea index (AI), the respiratory distress index (RDI) (p = 0.009) as well as a significant improvement in the degree of oxygen saturation (SaO2) (p = 0.008). The results were independent from the body mass since the patients did not lose weight during the follow-up period.

Domain swapping in inducible nitric-oxide synthase. Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer.

Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

Neuronal nitric-oxide synthase interaction with calmodulin-troponin C chimeras.

Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase.

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.

Analysis of neuronal NO synthase under single-turnover conditions: conversion of Nomega-hydroxyarginine to nitric oxide and citrulline.

Nitric oxide synthases (NOSs) are proposed to generate NO and citrulline from L-arginine in two steps: initial N-hydroxylation to generate Nomega-hydroxyarginine (NOHA) followed by a three-electron oxidation of the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation of O2. Studies done under multiple-turnover conditions suggest that 0.5 mol of NADPH is consumed to convert 1 mol of NOHA to products, implying that one electron from NADPH may be sufficient. To test this, we studied NOHA oxidation under single-turnover conditions using neuronal NOS (nNOS), whose heme iron reduction requires bound calmodulin. The heme iron in calmodulin-bound nNOS was reduced with excess NADPH under anaerobic conditions, calmodulin was then dissociated from nNOS to prevent subsequent heme iron reduction, NOHA was added, and the reaction initiated by exposure to air. Spectra obtained at each step were consistent with buildup of NOHA-bound ferrous nNOS prior to air exposure. Reactions containing graded amounts of nNOS produced L-citrulline in linear relation (1.2 +/- 0.1 mol of citrulline per mole of nNOS). Nitrite and nitrate also accumulated as NO-derived products. Control reactions that contained L-arginine instead of NOHA, no enzyme, or ferric nNOS did not generate products. Thus supplying a single electron from NADPH to the heme iron permits nNOS to catalyze one full round of citrulline and NO synthesis from NOHA upon exposure to O2. These data provide a molecular explanation for the NADPH requirement in the second step of the biosynthetic reaction, implicate ferrous-dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.

Incorporation of copper into the yeast Saccharomyces cerevisiae. Identification of Cu(I)--metallothionein in intact yeast cells.

Copper is an essential metal ion to many living organisms, including mammals, as it mediates a wide variety of important biochemical processes. At elevated concentrations, copper is extremely toxic to host cells. This paradoxical nature of copper has necessitated a highly regulated procedure for its cellular accumulation, transport, and excretion. One important group of proteins involved in eukaryotic copper speciation is the protein metallothionein. Luminescence microscopy data, emission, and circular dichroism spectral data are reported as copper is incorporated into metallothionein by the yeast Saccharomyces cerevisiae. These techniques provide information on the mechanism of copper uptake by S. cerevisiae. A two-stage kinetic mechanism for the uptake of copper from the growth medium by the yeast cells is observed. The first stage displays an uptake rate that is dependent on the initial copper concentration of the growth medium, and lasts for approximately 6 h. The second stage has a slower rate of copper uptake than the first, but the kinetics are independent of the initial copper concentration. Emission spectra recorded directly from the intact yeast cells (at 77 K) show that the cellular incorporation of copper proceeds via several species, eventually leading to storage of the copper in the form of Cu-metallothionein. The photomicrographs of yeast cells grown in a copper-containing medium clearly show an orange luminescence, indicating the formation of a Cu(I)-thiolate species. The identification of this species as copper-metallothionein was confirmed by measurement of the circular dichroism and emission properties following excretion and isolation of the copper-containing protein from the yeast cells. Analysis of the emission spectrum from S. cerevisiae Cu-metallothionein at 77 K reveals two emission bands, centered at 570 and 700 nm. The high-energy emission band exhibits a two-component decay, with excited state lifetimes of 4.70 and 48.5 microseconds. The low-energy emission exhibits one major decay component with a lifetime of 1.13 microseconds. A high-molecular-weight, copper-containing species is also isolated from the yeast cells and is characterized spectroscopically.

Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity.

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.

Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

Copper binding to rabbit liver metallothionein. Formation of a continuum of copper(I)-thiolate stoichiometric species.

Circular dichroism and ultraviolet absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(I)-thiolate binding clusters in rabbit liver metallothionein during addition of Cu+ to aqueous solutions of Zn7-metallothionein 2 and Cd5Zn2-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the alpha and beta domains. Spectral data which probe the distribution of Cu(I) between the two binding domains clearly show that both the site of binding (alpha or beta), and the structures of the specific metal-thiolate clusters formed, are dependent on temperature and on the nature of the starting protein (either Zn7-metallothionein or Cd5Zn2-metallothionein). CD spectra acquired during the addition of Cu+ to Zn7-metallothionein show that Cu+ replace the bound Zn(II) in a domain-distributed manner with complete removal of the Zn(II) after addition of 12 Cu+. Spectral and metal analyses prove that a series of Cu(I)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(I):metallothionein stoichiometries. Observation of a series of spectral saturation points signal the formation of distinct optically active Cu(I)-thiolate structures for the Cu9Zn2-metallothionein, Cu12-metallothionein, and the Cu15-metallothionein species. These data very clearly show that for Cu(I) binding to Zn7-metallothionein, there are several key Cu(I):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu12-metallothionein species are defined by bands located at 255(+) nm and 280(-) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data recorded between 3 degrees C and 52 degrees C as Cu+ is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temperature dependent. These changes in spectral properties are interpreted in terms of kinetic versus thermodynamic control of the metal-binding pathways as Cu+ binds to the protein. At low temperatures (3 degrees C and 10 degrees C) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu12-metallothionein. At higher temperatures (> 30 degrees C) the activation barrier is overcome, allowing formation of new Cu(I)-thiolate clusters with unique spectral properties, especially at the Cu9Zn2-metallothionein point. The CD spectra also show that a Cu15-metallothionein species with a well-defined, three-dimensional structure forms at all temperatures, characterized by a band near 335 nm, indicating the presence of diagonal Cu(I).(ABSTRACT TRUNCATED AT 250 WORDS)

Chiral copper(I)-thiolate clusters in metallothionein and glutathione.

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal-thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M7S20 structure are M4(Scys)11 (alpha domain) and M3(Scys)9 (beta domain). Up to 12 Cu(I) ions can displace the 7 Zn(2+) ions bound per molecule in Zn-MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu12S20 structure of Cu6(Scys)11 and Cu6(Scys)9 for the alpha and beta domains, respectively. The circular dichroism (CD) spectra recorded as Cu+ is added to Zn-MT to form Cu12-MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(i); because the Cu(I)-thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)-MT species. Model Cu(I)-thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)-thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)-thiolate clusters in the alpha and beta domains of Cu12-MT are proposed.

Spectroscopic studies of copper, silver and gold-metallothioneins.

Metallothionein is a ubiquitous protein with a wide range of proposed physiological roles, including the transport, storage and detoxification of essential and nonessential trace metals. The amino acid sequence of isoform 2a of rabbit liver metallothionein, the isoform used in our spectroscopic studies, includes 20 cysteinyl groups out of 62 amino acids. Metallothioneins in general represent an impressive chelating agent for a wide range of metals. Structural studies carried out by a number of research groups (using (1)H and (113)Cd NMR, X-ray crystallography, more recently EXAFS, as well as optical spectroscopy) have established that there are three structural motifs for metal binding to mammalian metallothioneins. These three structures are defined by metal to protein stoichiometric ratios, which we believe specifically determine the coordination geometry adopted by the metal in the metal binding site at that metal to protein molar ratio. Tetrahedral geometry is associated with the thiolate coordination of the metals in the M(7)-MT species, for M = Zn(II), Cd(II), and possibly also Hg(II), trigonal coordination is proposed in the M(11-12)-MT species, for M = Ag(I), Cu(I), and possibly also Hg(II), and digonal coordination is proposed for the metal in the M(17-18)-MT species for M = Hg(II), and Ag(I). The M(7)-MT species has been completely characterized for M = Cd(II) and Zn(II). (113)Cd NMR spectroscopic and x-ray crystallographic data show that mammalian Cd(7)-MT and Zn(7)-MT have a two domain structure, with metal-thiolate clusters of the form M(4)(S(cys))(11) (the alpha domain) and M(3)(S(cys))(9) (the beta domain). A similar two domain structure involving Cu(6)(S(cys))(11) (alpha) and Cu(6)(S(cys))(9) (beta) copper-thiolate clusters has been proposed for the Cu(12)-MT species. Copper-, silver- and gold-containing metallothioneins luminesce in the 500-600 nm region from excited triplet, metal-based states that are populated by absorption into the 260-300 nm region of the metal-thiolate charge transfer states. The luminescence spectrum provides a very sensitive probe of the metal-thiolate cluster structures that form when Ag(I), Au(I), and Cu(I) are added to metallothionein. CD spectroscopy has been used in our laboratory to probe the formation of species that exhibit well-defined three-dimensional structures. Saturation of the optical signals during titrations of MT with Cu(I) or Ag(I) clearly show formation of unique metal-thiolate structures at specific metal:protein ratios. However, we have proposed that these M=7, 12 and 18 structures form within a continuum of stoichiometries. Compounds prepared at these specific molar ratios have been examined by X-ray Absorption Spectroscopy (XAS) and bond lengths have been determined for the metal-thiolate clusters through the EXAFS technique. The stoichiometric ratio data from the optical experiments and the bond lengths from the XAS experiments are used to propose structures for the metal-thiolate binding site with reference to known inorganic metal-thiolate compounds.