Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Cytochromes c-550 (acidic), c-550 (basic), c-551 and c-552.5 from Thiobacillus versutus have been highly purified and characterized. Their spectral properties at 77 K are described. Oxidation-reduction titrations of cytochromes c-550 (acidic) and c-550 (basic) showed them to exhibit Nernst values of n = 1, with single redox centres in the cytochromes, and to have midpoint redox potentials at pH 7.0 (E_m,7) of 290 and 260 mV, respectively. Cytochrome c-551 contained two separately titratable redox components, each giving n = 1. The low potential centre (55% of titratable cytochrome) and the high potential centre (45%) had E_m,7 values of ?115 and +240 mV, espectively. Cytochrome c-552.5 also contained at least two redox centres. One (65% of titratable cytochrome) had n = 1 and E_m,7 = 220mV. The remaining 35% appeared to be a low potential component with an E_m,7 possibly as low as ?215 mV. the roles of these cytochromes in respiratory thiosulphate oxidation are discussed.     

Purification and characterization of two essential cytochromes of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus A2 (Thiobacillus versutus)

Four c-type cytochromes were purified by several procedures including chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephadex G-75, G-100 and G-200 and chromatofocusing. Cytochrome c-551 had a pI value of 5.2 and an M_r of 260 000 consisting of six non-covalently bound polypeptides each with an M_r of 43 000, and contained four to five haems. Cytochrome c-552.5 had a pI value of 4.8 and an M_r of 56 000 consisting of two polypeptides with the same M_r 29 000, and contained two haems. Cytochromes c-551 and c-552.5 were reduced by ascorbate to about 70 and 60% of the fully dithionite-reduced values, respectively, and both were essential components in the thiosulphate-oxidizing multi-enzyme system (other components of the system were ‘enzyme A’, ‘enzyme B’ and sulphite: cytochrome c oxidoreductase). These two cytochromes functioned as electron carriers and effectors in the oxidation of thiosulphate. Some evidence suggested that cytochrome c-551 might be a specialized electron transfer component for sulphonate-sulphur oxidation. Both cytochromes could be reduced by thiosulphate in the presence of enzymes A and B. Cytochrome c-550 (basic) and cytochrome c-550 (acidic) were small proteins with M_r 15 000 and 14 000 and pI values of over 8 and 5, respectively. Their physiological role is uncertain.     

Oxidation-reduction potentials of respiratory chain components in Thiobacillus A2

(1) Cells of Thiobacillus A2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the 'invisible', or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Transpositional Mutagenesis of Thiobacillus novellus and Thiobacillus versutus

Transpositional mutagenesis of Thiobacillus novellus by Tn501 was achieved by means of the incompatibility of IncP plasmids. Tn501 insertion caused three types of mutant phenotypes: isoleucine auxotrophy, lysine auxotrophy, and a reduced ability to oxidize reduced sulfur compounds and to fix CO₂. Oxidation rates for elemental sulfur (S⁰), thiosulfate (S₂O₃²⁻), and tetrathionate (S₄O₆²⁻) in mutants of the latter type were reduced relative to those of the nonmutant control strain. Incorporation of labeled bicarbonate (H¹⁴CO₃⁻) was also significantly impaired. Although suicide vehicles were not useful for the introduction of transposons into T. novellus, this method was effective for the Tn1721-induced mutagenesis of Thiobacillus versutus. Tn1721 insertions resulted in the loss of the natural resistance of T. versutus to arsenate and gentamicin and in auxotrophies for isoleucine-valine, arginine, phenylalanine, valine, and panthothenate. Transpositional mutagenesis by either method should prove to be a useful tool for further study of these and other members of the genus Thiobacillus.     

Oxidation-reduction potentials and absorption spectra of two b-type cytochromes from the halophilic archaebacterium, Halobacterium halobium

The oxidation-reduction midpoint potentials were determined for two b-type cytochromes, which had been solubilized from the membrane of Halobacterium halobium and partially purified. The two b-type cytochromes have oxidation-reduction midpoint potentials of 175 and 7 mV, respectively. These b-type cytochromes could also be resolved by difference absorption spectroscopy, which revealed one b-type cytochrome with absorption maximum (alpha-peak) at 558 nm, reducible by ascorbate-tetramethyl-p-phenylenediamine, and the other with absorption maximum (alpha-peak) at 560 nm, reducible by dithionite. Different substrates such as succinate, NADH, and alpha-glycerophosphate were used to study the b-type cytochromes in situ when bound to the membrane in a functional state. Reducing equivalents from succinate and alpha-glycerophosphate appear to enter the respiratory chain at the 175 mV b-type cytochrome. Cytochrome a3 is spectrophotometrically shown to be present in the membrane of H. halobium.     

A periplasmic location for the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus

Abstract Evidence is presented to show that the thiosulphate-oxidising multi-enzyme system from Thiobacillus versutus has a periplasmic location, and that the oxygen-binding site of the cytochrome oxidase ( aa ₃) is on the inner surface of the membrane. A scheme for the mechanism of generation of a proton motive force during electron flow from thiosulphate to oxygen via cytochrome c and aa ₃ is proposed.     

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.     

Oxidation-reduction potentials of cytochromes in Ascaris muscle mitochondria: high-redox-potential cytochrome b558 in complex II (succinate-ubiquinone reductase)

Oxidation-reduction midpoint potentials (Ems) were determined at pH 7.0 for cytochromes in the anaerobic respiratory chain of Ascaris mitochondria by redox titration techniques. Cytochrome b558, which is associated with complex II that functions as fumarate reductase in the terminal step of the respiratory chain, was shown to have an Em of -34 mV in the isolated complex II and -54 mV in mitochondria. These values are much higher than the value of Ascaris cytochrome b558. In contrast, Ems of cytochromes C + C1 and cytochrome b559.5 were determined in situ to be 235 mV and 78 mV, respectively, which are comparable to those of their mammalian counterparts.     

Preliminary evidence for the linear nature of Thiobacillus versutus pTAV2 plasmid

Thiobacillus versutus was shown to harbour pTAV2 plasmid with certain characteristics atypical for cccDNA. Preliminary evidence is presented that the plasmid may have the structure of a double-stranded linear DNA molecule.     

The structural homology of amicyanin from Thiobacillus versutus to plant plastocyanins

The complete amino acid sequence of the blue copper protein amicyanin of Thiobacillus versutus, induced when the bacterium is grown on methylamine, has been determined as follows: QDKITVTSEKPVAAADVPADAVVVGIEKMKYLTPEVTIKAGETVYWVNGEVMPHNVA FKKGIVGEDAFRGEMMTKDQAYAITFNEAGSYDYFCTPHPFMRGKVIVE. The four copper ligand residues in this 106-residue-containing polypeptide chain are His54, Cys93, His96, and Met99. The Thiobacillus amicyanin is 52% similar to the amicyanin of Pseudomonas AM1, the only other copper protein known with the same spacing between the second histidine ligand and the methionine ligand. T. versutus amicyanin contains no cysteine bridge and is more closely related to the plant copper protein plastocyanin than to the bacterial copper protein azurin. Alignment of the two known amicyanin sequences with the consensus sequence of the plastocyanins and comparison with the known three-dimensional structure of poplar leaves plastocyanin reveals that the bacterial proteins have the same overall structure with two beta-sheets packed face to face. The major structural differences between the amicyanins and the plastocyanins appear to be located in two of the five loops that connect the six identified beta-strands of the amicyanins. The first of these two loops, connecting strands F and G, contains a ligand histidine and must have a different conformation from the same loop in the plastocyanins because it is shorter by two amino acids. Further differences occur in the loop connecting the strands D and E. This loop contains only 17 residues in amicyanin whereas the corresponding loop of plastocyanin contains 25 residues. Despite these differences the amicyanins appear much closer related to the plastocyanins than to the azurins. The present findings demonstrate that the occurrence of blue copper proteins with clearly plastocyanin-like features is not restricted to photosynthetic redox chains.     

Purification and some properties of sulfite oxidase from Thiobacillus neapolitanus

Summary Sulfite oxidase has been purified 100-fold from extracts of Thiobacillus neapolitanus. The enzymatic activity, measured as the rate of oxygen consumption, was stimulated some 1.5–2-fold by 3.3mm AMP throughout the course of purificationm indicating that a single enzyme is responsible for the oxidation of sulfite in the presence and absence of AMP. Reduced glutathione inhibited activity and prevented the AMP-dependent rate stimulation. The rate of sulfite oxidation in the presence and absence of AMP was first order with respect to the sulfite concentration.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.Part of a dissertation submitted by W. P. Hempfling to Yale University in partial fulfillment of the requirements for the Ph. D. degree, 1964.     

Purification and some properties of thiosulphate-cleaving enzyme from Thiobacillus novellus

A thiosulphate-cleaving enzyme was purified from Thiobacillus novellus and some of its properties studied. The enzyme showed an absorption peak at 279 nm and no peaks between 300 and 650 nm. Its Mr was 38,000. Although the crude enzyme cleaved thiosulphate to form sulphite without addition of cyanide, the purified enzyme required cyanide to cleave thiosulphate. The Km values for thiosulphate and cyanide of the purified enzyme were 1.0 mM and 0.3 mM, respectively. One mol of the enzyme formed 10 mol of thiocyanate per s from thiosulphate and cyanide. The thiosulphate-cleaving activity of the enzyme was strongly inhibited by cysteine, while beta-mercaptoethanol was less inhibitory. The factor which accepted sulphur from thiosulphate in the crude preparation of thiosulphate-cleaving enzyme seemed to be a relatively labile compound with an Mr of 10,000 x 20,000.     

Chemolithotrophic ATP synthesis and NAD(P) reduction in Thiobacillus tepidarius and T. versutus

Thiosulphate-dependent reduction of NAD and NADP in intact cells of Thiobacillus tepidarius and T. versutus was severely inhibited or abolished by FCCP at concentrations that did not affect ATP synthesis over the same time scale. Thiosulphate-dependent ATP synthesis in T. tepidarius was abolished by the ATPase inhibitor DCCD, which did not affect NAD or NADP reduction at the concentrations tested. These results indicate that energy-dependent NAD(P) reduction using reversal of electron transfer from cytochrome b or c in thiobacilli is directly driven by the p generated by thiosulphate oxidation, and does not require the participation of ATP. While NAD(P) reduction and ATP synthesis are thus both effected by sulphur compound oxidation, there is no obligatory link between them.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - G-6-P glucose 6-phosphate - FCCP carbonylcyanide p-fluoromethoxyphenylhydrazone     

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

Purification, crystallization and preliminary X-ray investigation of quinoprotein methylamine dehydrogenase from Thiobacillus versutus

The enzyme methylamine dehydrogenase or primary-amine:(acceptor) oxidoreductase (deaminating) (EC 1.4.99.3) was purified from the bacterium Thiobacillus versutus to homogeneity, as judged by polyacrylamide gel electrophoresis. The native enzyme has a Mr of 123 500 and contains four subunits arranged in a alpha 2 beta 2 configuration, the light and heavy subunits having a Mr of 12900 and 47500 respectively. The isoelectric point is 3.9. The purified enzyme was crystallized from 37--42% saturated ammonium sulphate in 0.1 M sodium acetate buffer, pH 5.0. The space group is P3(1)21 or P3(2)21, with one alpha 2 beta 2 molecule in the asymmetric unit. The cell dimensions are: a = b = 13.01 nm; c = 10.40 nm. The X-ray diffraction pattern extends to at least 0.25-nm resolution.