The Cu_A Center of a Soluble Domain from Thermus Cytochrome ba_3. An NMR Investigation of the Paramagnetic Protein

The Cu_A center in subunit II of cytochrome c oxidase, the terminal enzyme of aerobic respiration, transfers electrons from cytochrome c to the proton-pumping machinery in subunit I. The unique electronic absorption and EPR spectra of Cu_A exclude it from classification with the well-studied biological copper centers. High-resolution X-ray structures of Cu_A-containing proteins reveal two copper atoms approximately 2.5 Å apart, bridged by two cysteine sulfurs. Each Cu has a terminal histidine ligand and a weak ligand, methionine for one and a main chain carbonyl for the other. These structures are consistent with earlier EPR measurements and theoretical calculations, which predicted a highly delocalized mixed-valence [Cu(II),Cu(I)] Cu_A site. Here we report ^1H NMR measurements at 600 MHz on a soluble Cu_A domain from Thermus thermophilus cytochrome ba_3

Effect of Redox State on the Folding Free Energy of a Thermostable Electron-Transfer Metalloprotein: The Cu_A Domain of Cytochrome Oxidase from Thermus thermophilus

The unfolding of the Cu_A domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)^1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 °C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of −65 kJ/mol. The corresponding value for the oxidized protein (−85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide

Effect of Redox State on the Folding Free Energy of a Thermostable Electron-Transfer Metalloprotein: The Cu_A Domain of Cytochrome Oxidase from Thermus thermophilus

The unfolding of the Cu_A domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)^1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 °C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of −65 kJ/mol. The corresponding value for the oxidized protein (−85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide

Electrochemistry of the Cu_A domain of Thermus thermophilus cytochrome ba _3

The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba _3 has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)_2Cu]^(+/0) electrode reaction were determined by variable-temperature electrochemistry (ΔS°_(rc) = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)_2Cu]^(+/0) electron transfer. An irreversible oxidation of [Cu(Cys)_2Cu]^+ at 1 V vs NHE confirms that the Cu^(II):Cu^(II) state of Cu_A is significantly destabilized relative to the Cu^(II) state of analogous blue-copper proteins

Water-Soluble, Recombinant Cu_A-Domain of the Cytochrome ba_3 Subunit II from Thermus thermophilus

Recently, the genes of cytochrome ba_3 from Thermus thermophilus [Keightley, J. A., et al. (1995) J. Biol. Chem. 270, 20345−20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (Cu_A) domain of cytochrome ba_3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The Cu_A-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the Cu_A site in cytochrome ba_3 is a binuclear center. The absorption spectrum of the Cu_A site, free of the heme interference in cytochrome ba_3, is similar to the spectra of other soluble fragments from the aa_3-type oxidase of Paracoccus denitrificans [Lappalainen, P., et al. (1993) J. Biol. Chem. 268, 26416−26421] and the caa_3-type oxidase of Bacillus subtilis [von Wachenfeldt, C., et al. (1994) FEBS Lett. 340, 109−113]. There are intense bands at 480 nm (3100 M^(-1) cm^(-1)) and 530 nm (3200 M^(-1) cm^(-1)), a band in the near-IR centered at 790 nm (1900 M^(-1) cm^(-1)), and a weaker band at 363 nm (1300 M^(-1) cm^(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly β-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa_3 molecule [Iwata, S., et al. (1995) Nature 376, 660−669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 92, 11955−11959]. However, the thermostability of the fragment described here (T_m ≈ 80 °C) and the stable binding of copper over a broad pH range (pH 3−9) suggest this protein may be uniquely suitable for detailed physical-chemical study

Expression of receptor tyrosine kinases epidermal growth factor receptor and HER-2/ neu in synovial sarcoma Presented, in part, at the 7th Connective Tissue Oncology Society Meeting, West Palm Beach, Florida, November 1–3, 2001.

BACKGROUND Synovial sarcomas are high-grade soft tissue neoplasms often characterized by a biphasic spindle and epithelioid cell morphology. The majority of synovial sarcomas harbor a specific chromosomal translocation in which the proximal portion of the SYT gene at chromosome 18q11 is fused to the distal portion of one of several duplicated SSX genes (most notably SSX1 and SSX2 ) at chromosome Xp11. SYT / SSX1 translocations are seen in nearly three times as many synovial sarcomas as SYT / SSX2 translocations. Although the SYT / SSX2 fusion is usually associated with the monophasic disease pattern, the SYT / SSX1 fusion is present in tumors with biphasic or monophasic patterns. The SYT / SSX1 fusion gene is associated with more aggressive tumor growth and poor outcome. Despite advances in the therapy of local disease, distant metastasis remains the predominant cause of death. Accordingly, there is a need for alternate therapies, such as those recently developed against the receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and HER-2/ neu . METHODS Archival specimens of synovial sarcoma ( n = 38) representing 30 patients were assessed for EGFR and HER-2/ neu protein expression by standard immunohistochemical techniques. To validate the immunohistochemistry results, quantitative real-time polymerase chain reaction (Q-PCR) assays using either fresh and/or archival material was performed. The presence of gene amplification was determined by chromogenic in-situ hybridization. RESULTS EGFR and HER-2/ neu protein were detected by immunohistochemistry in 21 of 38 (55.3%) and 20 of 38 (52.6%) synovial specimens, respectively. EGFR immunoreactivity showed a granular and membranous pattern, whereas HER-2/ neu immunoreactivity demonstrated only a membrane pattern. Coexpression was observed in 13 of 38 specimens (34.2%). HER-2/ neu expression by immunohistochemistry in synovial sarcomas was restricted to tumors with the SYT / SSX1 translocations. Of 6 specimens with SSX2 translocation, none (0%) showed HER-2/ neu immunoreactivity and 1 (17%) demonstrated EGFR expression. Q-PCR demonstrated the presence of mRNA for EGFR and HER-2 / neu in 19 of 30 specimens (63.3%) and 22 of 30 specimens (73.3%), respectively. EGFR and HER-2 / neu were expressed at low concentrations compared with the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). No evidence of gene amplification was observed. CONCLUSIONS EGFR and HER-2/ neu are expressed in the majority of patients with SYT / SSX1 synovial sarcomas, albeit at low levels. Treatment with tyrosine kinase inhibitors may represent appropriate alternate therapy for these patients. Cancer 2005. © 2005 American Cancer Society.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34393/1/20847_ftp.pd

Electron-transfer studies with the CuA domain of Thermus thermophilus cytochrome ba_3

Flash photolysis has been used to initiate electron transfer from excited tris(2,2′-bipyridyl)ruthenium(II) to the Cu_A site of a soluble domain from subunit II of Thermus thermophilus cytochrome ba_3. Luminescence quenching of the excited state of the ruthenium(II) complex was observed at low protein concentrations (20–200 μM Cu_A domain), with second-order rate constants of 2.9 × 10^9 and 1.3 × 10^9 M^(−1) s^(−1) at low and high ionic strength, respectively. Transient absorption measurements demonstrate that 10–20% of the quenching arises from electron transfer (ET). At high protein concentrations (>250 μM Cu_A) and low ionic strength (5 mM Tris, pH 8.1), the quenching rate saturates due to ground-state complex formation; a first-order rate constant of 1.5 × 10^5 s^(−1) was estimated for ET in the complex. Given the high driving forces involved (ΔG° = 1.1 eV), it is possible that these ET reactions occur in the inverted driving-force regime. Spectroscopic measurements indicate that the T. thermophilus Cu_A domain and horse heart cytochrome c form a complex at low ionic strength, with an apparent dissociation constant K_d ∼ 5 μM