Characterization of cytochrome c-550 from cyanobacteria

Cytochrome c-550 has been purified from several cyanobacteria. It is a low-potential, auto-oxidizable cytochrome. This cytochrome should not be confused with a degradation product of cytochrome ? which may be formed during the isolation of the latter protein. Cytochromes c-550 are distinctive in size, amino-acid composition and N-terminal amino-acid sequence.     

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.     

Some properties of adenylate kinase from chemolithotrophically grown Thiobacillus novellus

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus novellus was purified 54-fold. The preparation was, on the basis of densitometer scans of polyacrylamide gels, at least 85% pure. The optimum pH in the forward direction (2 ADP ATP+AMP) was about 8.7, and in the reverse 8.2 The enzyme was specific for AMP, ADP and ATP with apparent K _m values of 0.04, 0.34 and 0.09 mM respectively. A double reciprocal plot of specific activity vs. (ADP)² was linear. Both AMP and ATP inhibited the forward reaction with AMP the more inhibitory of the two. AMP inhibition was competitive with respect to ADP with a K _i of 0.125 mM. Although Mg²⁺ was necessary for maximal activity, about 20% of this was obtained in its absence. Co²⁺ and Mn²⁺ at similar concentrations gave 46% and 26% respectively of the activity found with Mg²⁺. Apparent K _m for Mg²⁺ was about 0.054 mM in the forward and 0.15 mM in the reverse direction. pHMB and HgCl₂ were potent inhibitors. Inhibition by pHMB but not HgCl₂ was reversible by GSH or cysteine. Arrhenius plots gave an E _a of 3.25 kcal/mole/degree C in the forward direction without discontinuity. In the reverse, there was a break at 26.7°C with an E _a of 3.62 kcal/mole/degree C for lower temperatures and 3.92 kcal/mole/degree C for higher temperatures.Molecular weights of the enzyme were 46,300±300 by SDS PAGE and 47,800±200 by SDS PAGE after treatment with 5 M urea and about 40,000 by sucrose density gradient centrifugation.Abbreviations APS adenosine-5-phosphosulfate - DEAE diethylaminoethyl - DTT dithiothreitol - E_a energy of activation - ECTEOLA epichlorohydrin triethanolamine - G6P glucose-6-phosphate - GSH glutathione (reduced) - PAGE Polyacrylamide gel electrophoresis - pHMB parahydroxymercuribenzoate - PEP phosphoenolpyruvate - PPi inorganic pyrophosphate - SDS sodium dodecyl sulfate - TEAE triethylaminoethyl Supported by an operating grant to A.M.C. from NSERCSummer student research trainee     

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.     

A reassessment of the structure of Paracoccus cytochrome c-550

An amino acid sequence and a three-dimensional structure of cytochrome c-550 from the facultatively denitrifying aerobic bacterium Paracoccus denitrificans have been reported (Timkovich et al., 1976; Timkovich &; Dickerson, 1976). The amino acid sequence showed considerable similarity to Rhodospirillaceae (purple phototrophic bacterial) cytochrome c₂, but also had some unexpected features. We have reexamined the amino acid sequence and have found five discrepancies. The molecule contains an additional tryptophan residue, which was not detected in either the 2.5 Å crystallographic analysis or the original sequence investigation.     

1H NMR spectroscopy of Paracoccus denitrificans cytochrome c-550

The 1H NMR spectra of ferri- and ferro-cytochrome c-550 from Paracoccus denitrificans (ATCC 13543) have been investigated at 300 MHz. The ferri-cytochrome c-550 shows hyperfine-shifted heme methyl resonances at 29.90, 29.10, 16.70, and 12.95 ppm and a ligand methionyl methyl resonance at -15.80 ppm (pH 8 and 23 degrees C). Four pH-linked structural transitions were detected in spectra taken as a function of pH. The transitions have been interpreted as loss of the histidine heme ligand (pK less than or equal to 3), ionization of a buried heme propionate (pK = 6.3 +/- 0.2), displacement of the methionine heme ligand by a lysyl amino group (pK congruent to 10.5), and loss of the lysyl ligand (pK greater than or equal to 11.3). The temperature behavior of hyperfine-shifted resonances was determined. Two heme methyl resonances (at 16.70 and 12.95 ppm) showed downfield hyperfine shifts with increasing temperature. The cyanoferricytochrome had methyl resonances at 23.3, 20.1, and 19.4 ppm. NMR spectroscopy did not detect the formation of a complex with azide. The second-order rate constant for electron transfer between ferric and ferrous forms was determined to be 1.6 X 10(4) M-1 s-1. Heme proton resonances were assigned in both oxidation states by cross-saturation and nuclear Overhauser enhancement experiments. Spin-coupling patterns in the aromatic region of the ferro-cytochrome spectrum were investigated.     

Growth factor requirement of Thiobacillus novellus

Thiobacillus novellus cannot be grown in mineral salts media unless supplied with yeast extract. The requirement is only for miniscule amounts of yeast extract and is not fully expressed unless cells grown in a complex medium are allowed to multiply in a mineral salts medium for four to five generations. Individual sulfur-containing organic compounds, namely biotin, coenzyme A, and lipoic acid, but not reduced inorganic sulfur compounds, can substitute for the yeast extract requirement. Biotin can fully satisfy this requirement at a concentration insufficient to fulfill the biosynthetic sulfur needs; further, the organisms continue to incorporate ³⁵SO₄ into cellular protein in the presence of yeast extract or biotin. It is concluded that biotin is required as a growth factor and not owing to an inability to obtain sulfur from sulfate; the reasons why coenzyme A and thiamine pyrophosphate can substitute for biotin are discussed.Non-standard Abbreviations MS Mineral Salts Base     

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.     

The complete amino acid sequence of Nitrobacter agilis cytochrome c-550

The amino acid sequence of cytochrome c-550 from the chemoautotroph, Nitrobacter agilis, was completed by using solid-phase sequencing and conventional procedures. The cytochrome was composed of 109 amino acid residues and its molecular weight was calculated to be 12375 including haem c. The cytochrome was homologous to eukaryotic cytochromes c and some photosynthetic bacterial cytochromes c2. In particular, its primary structure was very similar to that of Rhodopseudomonas viridis cytochrome c2. Some of its properties were compared with those of other cytochromes c on the basis of the primary structure.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

The peroxidase activity of cytochrome c-550 from Paracoccus versutus.

Next to their natural electron transport capacities, c-type cytochromes possess low peroxidase and cytochrome P-450 activities in the presence of hydrogen peroxide. These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics. This study reports on the peroxidase activity of cytochrome c-550 from Paracoccus versutus and the loss of this activity in presence of H2O2. The rate-determining step in the peroxidase reaction of cytochrome c-550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron. The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H2O2, and the lack of a base catalyst such as the distal His of the peroxidases. This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 M H2O2. Steady-state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build-up of amino-acid radicals on the protein. The inactivation kinetics in the absence of reducing substrate are mono-exponential and shown to be concurrent with haem degradation up to 25 mM H2O2 (pH 8.0). At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H2O2, involving the formation of superoxide and ferrocytochrome c-550.     

The amino acid sequence of the cytochrome c-554(547) from the chemolithotrophic bacterium Thiobacillus neapolitanus.

An amino acid sequence is proposed for the cytochrome c-554(547) from the bacterium Thiobacillus neapolitanus N.C.I.B. 8539). It consists of a polypeptide chain of 91 residues, with a pair of haem-attachment cysteine residues at positions 15 and 18. There is similarity in sequence with each of the halves of the sequence of the dihaem cytochromes c4 and with a cytochrome c-554(548) from a halophilic strain of Paracoccus. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50127 (11 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.     

Growth of Thiobacillus novellus on mixed substrates (mixotrophic growth).

In a mixotrophic environment, Thiobacillus novellus concurrently utilized glucose and thiosulfate but showed no stimulation of growth rate or yield. In most mixotrophic environments examined, the growth rate was lower than the heterotrophic growth rate, the extent of the decrease depending on the concentration and relative proportion of thiosulfate and glucose in the medium. Both thiosulfate and glucose were degraded to their most oxidized products in mixotrophic medium, yet the biomass production in this medium was comparable to that found in heterotrophic medium containing glucose alone at the corresponding concentration. It was postulated that in mixotrophic medium the oxidation of thiosulfate, glucose, or partially that of both was uncoupled from energy generation. These results differ in many respects from those reported earlier by LeJohn et al. (J. Bacteriol. 94: 1484--1491, 1967); experiments designed to exactly duplicate some of the growth conditions employed by these workers did not resolve the discrepancy.     

Thiobacillus ferrooxidans cytochrome c-552: purification and some of its molecular features

Soluble cytochrome c-552 was purified from Thiobacillus ferrooxidans to an electrophoretically homogeneous state. The cytochrome showed absorption peaks at 276, 411 and 523 nm in the oxidized form and peaks at 315, 417, 523 and 552 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 13,800 on the basis of the amino acid composition and heme content, and 14,000 from SDS-polyacrylamide gel electrophoresis analysis. Its midpoint redox potential at pH 7.0 was determined to be +0.36 V. The N-terminal amino acid sequence of the cytochrome was determined as follows: A-G-G-A-G-G-P-A-P-Y-R-I-S-?-D-?-M-V-?-S-G-M-P-G-. Ferrocytochrome c-552 was oxidized by the membrane fraction of T. ferrooxidans, and the oxidation rate was more rapid at pH 3.0 than at pH 6.5. Ferricytochrome c-552 was reduced by Fe(II)-cytochrome c oxidoreductase with Fe2+ at pH 3.5, while horse ferricytochrome c was not reduced by the enzyme under the same reaction conditions.