Donita Sanders

CLONING AND EXPRESSION IN ESCHERICHIA COLI OF THE CYTOCHROME C552 GENE FROM THERMUS THERMOPHILUS HB8 : EVIDENCE FOR GENETIC LINKAGE TO AN ATP-BINDING CASSETTE PROTEIN AND INITIAL CHARACTERIZATION OF THE CYCA GENE PRODUCTS

We report sequence of Thermus thermophilus HB8 DNA containing the gene (cycA) for cytochrome c 552 and a gene (cycB) encoding a protein homologous with one subunit of an ATP-binding cassette transporter. The cycA gene encodes a 17-residue N-terminal signal peptide with following amino acid sequence identical to that reported by (Titani, K., Ericsson, L. H., Hon-nami, K., and Miyazawa, T. (1985) Biochem. Biophys. Res. Commun. 128, 781–787). A modified cycA was placed under control of the T7 promoter and expressed in Escherichia coli. Protein identical to that predicted from the gene sequence was found in two heme C-containing fractions. Fraction rC 552, characterized by an α-band at 552 nm, contains ∼60–70% of a protein highly similar to native cytochromec 552 and ∼30–40% of a protein that contains a modified heme. Cytochrome rC 552 is monomeric and is an excellent substrate for cytochromeba 3. Cytochrome rC 557is characterized by an α-band at 557 nm, contains ∼90% heme C and ∼10% of non-C heme, exists primarily as a homodimer, and is essentially inactive as a substrate for cytochromeba 3. We suggest thatrC 557 is a “conformational isomer” ofrC 552 having non-native, axial ligands to the heme iron and an “incorrect” protein fold that is stabilized by homodimer formation.

Electrochemistry of the Cu_A domain of Thermus thermophilus cytochrome ba _3

Abstract The electrochemistry of a water-soluble fragment from the CuA domain of Thermus thermophilus cytochrome ba3 has been investigated. At 25  °C, CuA 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 CuII:CuII state of CuA is significantly destabilized relative to the CuII state of analogous blue-copper proteins.

High-potential states of blue and purple copper proteins

Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.

Electron-transfer studies with the CuA domain of Thermus thermophilus cytochrome ba3

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.

Reaction of mercurials with the CuA center in the soluble fragment of cytochrome ba3 subunit II from Thermus thermophilus

Abstract Optical absorption, EPR and electrospray ionization mass spectrometries were used to characterize a stoichiometric reaction between mercurials and the soluble ba3–CuA protein from Thermus thermophilus. Either one Hg(II) or two RHg(II)ions react(s) to destroy the unique spectral properties of the CuA center. EPR spectra of the resulting product indicate that one Cu from the binuclear CuA center is released into the medium as a Type 2 Cu(II) while the other remains EPR silent. Mass spectra indicate that either one Hg(II) or two RHg(II) ions remain(s) bound to the protein along with one Cu, which is assumed to be a Cu(I) ion. The latter is slowly released from the protein under aerobic conditions as additional Type 2 Cu(II), and this process is catalyzed by fungal laccase, which serves as a strong one-electron oxidant.