Amino acid sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans

Rusticyanin is a small blue copper protein isolated from Thiobacillus ferrooxidans. The amino acid sequence of the rusticyanin has been determined by the structural characterization of tryptic and endoproteinase Asp-N peptides with use of amino terminal microsequencing, fast atom bombardment mass spectrometry, and electrospray triple-quadrupole mass spectrometry techniques. Amino acid analysis, carboxy-terminal sequence analysis, and circular dichroism spectroscopy were also performed on the protein. Amino acid sequence identity among rusticyanin and six other small blue copper proteins is apparent only in the limited C-terminal region of each protein bearing three of the four putative copper ligands. A structural model of the rusticyanin is proposed where the protein is principally a beta-barrel comprised of six strands. This model is consistent with the circular dichroism data and computational predictions of the secondary structure of rusticyanin. A feature of the model is the hypothesis that Asp 73 may serve as a fourth copper ligand.     

Isolation and characterization of a blue copper protein from Thiobacillus versutus

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1.     

Purification and some properties of a blue copper protein from Methylobacillus sp. strain SK1 DSM 8269

A blue protein was purified from the Methylobacillus sp. strain SK1 that is grown on methanol in the presence of copper ion. This protein was found to be a monomer with a molecular weight of 13,500. The Isoelectric point of the protein was estimated to be 8.8. The spectrum of the protein that was treated with ferricyanide showed a broad peak around 620 nm, but that of the dithionite-treated protein revealed no peaks. It contained 0.83 mol of EDTA-stable copper per mol protein. Under air, the protein accelerated the inactivation of methanol dehydrogenase (MDH). The protein was reducible by phenazine methosulfate or by active MDH that was prepared from cells that were grown in the absence of added copper, but not by methanol, dichlorophenol indophenol, or inactive MDH that was prepared from cells that were grown in the presence of added copper. It was also reducible by active MDH in the presence of methanol. The absorption peak at 340 nm of the active MDH disappeared after the enzyme was treated with ferricyanide, hydrogen peroxide, or the purified blue protein. The inactive MDH also showed no peak at 340 nm. The 340-nm peak was not recovered after incubation of the inactive MDH and blue protein-treated active MDH with dithionite or methanol. The inactive MDH and blue protein-treated active MDH co-migrated with the active MDH preparation on nondenaturing polyacrylamide gel, and contained two non-identical subunits with molecular weights that were identical to those of the active MDH. The N-terminal amino acid sequence of the protein was Ala-Gly-Cys-Ser-Val-Asp-Val-Glu-Ala-Asn-Asp-Ala-Met-Gln-Phe. An analysis of the amino acid composition revealed that the protein contained no tryptophan. It contained three cysteines per mol protein. The blue protein in Methylobacillus sp. strain SK1 was produced only in the cells that were grown in the copper-supplemented medium.     

Effect of divers anions on the electron-transfer reaction between iron and rusticyanin from Thiobacillus ferrooxidans

Rusticyanin is a soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing chemolithotrophically on soluble ferrous sulfate. The one-electron-transfer reactions between soluble iron and purified rusticyanin were studied by stopped-flow spectrophotometry in acidic solutions containing each of 14 different anions. The second-order rate constants for both the Fe(II)-dependent reduction and the Fe(III)-dependent oxidation of the rusticyanin varied as a function of the identity of the principal anion in solution. Analogous electron-transfer reactions between soluble iron and bis(dipicolinato)cobaltate(III) or bis(dipicolinato)ferrate(II) were studied by stopped-flow spectrophotometry under solution conditions identical with those of the rusticyanin experiments. Similar anion-dependent reactivity patterns were obtained with soluble iron whether the other reaction partner was rusticyanin or either of the two organometallic complexes. The Marcus theory of outer-sphere electron transfer reactions was applied to this set of kinetic data to demonstrate that the rusticyanin may possess at least two electron-transfer pathways for liganded iron, one where the pattern of electron-transfer reactivity is controlled largely by protein-independent activation parameters and one where the protein exhibits an anion-dependent kinetic specificity. The exact role of rusticyanin in the iron-dependent respiratory electron transport chain of T. ferrooxidans remains unclear.     

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.     

Inhibition of iron(II) oxidation by arsenic(III,V) in Thiobacillus ferrooxidans: Effects on arsenopyrite bioleaching

Summary The more complex inhibitory effect of As(III) than that of As(V) on Fe(II) oxidation in a non-growing Thiobacillus ferrooxidans suspension was demonstrated. The yield of arsenic bioextraction from a chalcopyrite concentrate was not affected by arsenic inhibition due to the low sensitivity of the strain to arsenic ions, supported by a spontaneous conversion of As(III) to As(V).     

Sulfate-dependent iron oxidation by Thiobacillus ferrooxidans: characterization of a new EPR detectable electron transport component on the reducing side of rusticyanin

Iron(II) oxidation by pH 2.5 HCl-washed cells of Thiobacillus ferrooxidans is known to be sulfate dependent. Sulfate dependence of the autooxidation of a novel component in the electron transport pathway is demonstrated. This component exhibits an electron paramagnetic resonance (EPR) signal in the oxidized state at g = 2.005 distinguishable from the g = 2.08 signal attributed to rusticyanin. The novel component is proposed to be a three-iron-sulfur cluster based upon the g value, lineshape, and temperature dependence. Oxyanion specificity for the EPR signal has the same dependence on sulfate as does iron(II) oxidation. By using azide to inhibit electron transfer to oxygen, sulfate was shown to be involved in electron transfer from the g = 2.005 component to the copper of rusticyanin.     

Backbone dynamics of rusticyanin: the high hydrophobicity and rigidity of this blue copper protein is responsible for its thermodynamic properties

Local dynamics and solute-solvent exchange properties of rusticyanin (Rc) from Thiobacillus ferrooxidans have been studied by applying heteronuclear ((1)H, (15)N) NMR spectroscopy. (15)N relaxation parameters have been determined for the reduced protein, and a model-free analysis has been applied. The high average value of the generalized order parameter, S(2) (0.93), indicates that Rc is very rigid. The analysis of cross correlation rates recorded in both the reduced and the oxidized forms conclusively proves that Rc possesses the same dynamic features in both oxidation states. The accessibility of backbone amide protons to the solvent at different time scales has also been studied by applying specific heteronuclear pulse sequences and by H(2)O/D(2)O exchange experiments. These experiments reveal that rusticyanin is extremely hydrophobic. The first N-35 amino acids, not present in the other BCPs, protect the beta-barrel core from its interaction with the solvent, and thus, this is one of the main factors contributing to the hydrophobicity. Both characteristics (high rigidity and hydrophobicity) are maintained in the metal ion surroundings.     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

The syntheses and properties of cobalt(II), nickel(II) and copper(II) complexes with some heterocyclic dithiocarbamates

New cobalt(II), Nickel(II) and copper(II) dithiocarbamato complexes of the type M(Rdtc)₂ (Rdtc = 4-phenylpiperidinedithiocarbamate and N-phenylpiperazinedithiocarbamate) have been prepared and characterized through elemental analyses, conductivity measurements, spectral (electronic and IR) studies, magnetic moment measurements at different temperatures, e.p.r. techniques and thermal analyses (TG and DTG). The dithioligands exhibit bidentate behaviour in all the complexes. The magnetic moments studies suggest that there is no significant interaction between copper ions, and the e.p.r. data provide parameters typical of sulphur coordination in planar CuS₄ chromophores.     

An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role

When grown on methylamine as a sole carbon source, Paracoccus denitrificans synthesizes a Type I blue copper protein which mediates electron transfer between methylamine dehydrogenase and cytochrome c. This blue copper protein does not serve as an electron acceptor for methanol dehydrogenase and is not synthesized by cells grown on methanol or succinate. The blue copper protein and methylamine dehydrogenase were localized in the periplasm of P. denitrificans, whereas formate dehydrogenase was cytoplasmic. The copper protein can be purified to high yield in a single step from the periplasmic subcellular fraction prepared from P. denitrificans. The purified protein contains a single 15,000-Da polypeptide chain and one copper atom/molecule and exhibits a pI of 4.8. The oxidized form of the protein absorbs strongly at 595 nm and weakly at 464 nm. The physical and physiological properties of this protein indicate that it is not an azurin, but representative of another class of blue copper proteins.     

Purification and some properties of cytochrome c-552 from a sulfur-oxidizing bacterium, Thiobacillus thiooxidans.

A soluble cytochrome c-552 from Thiobacillus thiooxidans was highly purified and its physico-chemical properteis were studied. The absorption maxima were at 552,523,418 nm in the reduced from and at 412 nm in the oxidized form. The pyridine hemochrome spectrum was the same as that of other cytochromes c. The molecular weight, estimated by the gel filtration method, was found to be 12,600. The isoelectric point was determined to be 9.2-9.3 by the electrofocusing technique. The standard oxidation-reduction potential of this cytochrome was +0.247 V.     

Antimicrobial and mutagenic activity of some carbono- and thiocarbonohydrazone ligands and their copper(II), iron(II) and zinc(II) complexes.

Several mono- and bis- carbono- and thiocarbonohydrazone ligands have been synthesised and characterised; the X-ray diffraction analysis of bis(phenyl 2-pyridyl ketone) thiocarbonohydrazone is reported. The coordinating properties of the ligands have been studied towards Cu(II), Fe(II), and Zn(II) salts. The ligands and the metal complexes were tested in vitro against Gram positive and Gram negative bacteria, yeasts and moulds. In general, the bisthiocarbonohydrazones possess the best antimicrobial properties and Gram positive bacteria are the most sensitive microorganisms. Bis(ethyl 2-pyridyl ketone) thiocarbonohydrazone, bis(butyl 2-pyridyl ketone)thiocarbonohydrazone and Cu(H2nft)Cl2 (H2nft, bis(5-nitrofuraldehyde)thiocarbonohydrazone) reveal a strong activity with minimum inhibitory concentrations of 0.7 microgram ml-1 against Bacillus subtilis and of 3 micrograms ml-1 against Staphylococcus aureus. Cu(II) complexes are more effective than Fe(II) and Zn(II) ones. All bisthiocarbono- and carbonohydrazones are devoid of mutagenic properties, with the exception of the compounds derived from 5-nitrofuraldehyde. On the contrary a weak mutagenicity, that disappears in the copper complexes, is exhibited by monosubstituted thiocarbonohydrazones.     

The production of hydroxyl radical from copper(I) complex systems of bleomycin and tallysomycin: comparison with copper(II) and iron(II) systems.

In contrast with BLM(or TALM)-Cu(II) complex system, Cu(I)-O₂ system of BLM(or TALM) as well as the corresponding Fe(II) system evidently produces reactive oxygen radicals as detected by ESR spin trapping. The sulfhydryl compound strongly prevented the generation of hydroxyl radical in BLM(or TALM)-Cu(I)-O₂ system. TALM forms metal complexes similar to BLM. The action mechanism of BLM and TALM has been proposed to be substantially same.     

Potential of Thiobacillus ferrooxidans for waste gas purification. Part 1. Kinetics of continuous ferrous iron oxidation

Kinetic data of ferrous iron oxidation by Thionacillus ferrooxidans were determined. The aim was to remove H₂S (<0.5 ppm) from waste gas by a process proposed earlier. Kinetic data necessary for industrial scale-up were investigated in a chemostat airlift reactor (dilution rate 0.02–0.12 h^–1; pH 1.3). Due to the low pH, ferric iron precipitation and wall growth could be avoided. The maximum ferrous iron oxidation rate of submersed bacteria was 0.77 g 1^–1 h^–1, the maximum specific growth rate about 0.12 h^–1 and the yield coefficient was found to be 0.007 g g^–1 Fe²⁺. The specific O₂ demand of an exponentially growing, ironoxidizing batch culture was 1.33 mg O₂ mg^–1 biomass h^–1. The results indicate that a pH of 1.3 has no negative influence on the kinetics of iron oxidation and growth. Correspondence to: W. Schäfer-Treffenfeldt     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate