Thermostable peroxidase from Bacillus stearothermophilus

A peroxidase from Bacillus stearothermophilus was purified to homogeneity. The enzyme (Mr 175,000) was composed of two subunits of equal size, and showed a Soret band at 406 nm. On reduction with sodium dithionite, absorption at 434 nm and 558 nm was observed. The spectrum of reduced pyridine haemochrome showed peaks at 418, 526 and 557 nm; the reduced minus oxidized spectrum of pyridine haemochrome showed peaks of 418, 524 and 556 nm with a trough at 452 nm. These results indicate that the enzyme contained protohaem IX as a prosthetic group. The optimum pH was about 6 and the apparent optimum temperature was 70 degrees C. The enzyme was relatively stable up to 70 degrees C; at 30 degrees C it was stable for a month. The enzyme had peroxidase activity toward a mixture of 2,4-dichlorophenol and 4-aminoantipyrine with a Km for H2O2 of 1.3 mM. It also acted as a catalase with a Km for H2O2 of 7.5 mM.     

Purification and characterization of catalase from a facultative alkalophilic Bacillus

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).     

Molecular and enzymatic properties of "cytochrome aa3"-type terminal oxidase derived from Nitrobacter agilis

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Nitrobacter agilis to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed absorption peaks at 422, 598, and 840 nm in the oxidized form, and at 442 and 606 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 436 and 604 nm, and the latter peak had a shoulder at 599 nm. The enzyme possessed 1 mol of heme a and 1.6 g-atom of copper per 41,000 g, and was composed of two kinds of subunits of 51,000 and 31,000 daltons. These results show that the structurally minimal unit of the enzyme molecule is composed of one molecule each of the two subunits and contains 2 molecules of heme a and 2-3 atoms of copper. the enzyme rapidly oxidized ferrocytochromes c of several eukaryotes as well as N. agilis ferrocytochrome c-552. The reactions catalyzed by the enzyme were strongly inhibited by KCN. The reduction product of oxygen catalyzed by the enzyme was concluded to be water on the basis of the ratio of ferrocytochrome c oxidized to molecular oxygen consumed.     

One of two copper atoms is not necessary for the cytochrome c oxidase activity of Pseudomonas AM 1 cytochrome aa3

From Pseudomonas AM 1 grown in a medium deficient in Cu, aa3-type cytochrome c oxidase was purified which contained 2 molecules of haem a and one atom of Cu per molecule. The enzyme showed absorption peaks at 428 and 595 nm in the oxidized form and at 442 and 604 nm in the reduced form, and its CO complex showed peaks at 432 and 602 nm. The enzyme in the oxidized state showed an obscure absorption peak around 800 nm instead of a peak at 820 nm. One mol of the enzyme oxidized maximally 76, 75, and 98 mol of the ferrocytochromes c of Candida krusei, horse and Pseudomonas AM 1 per sec, respectively. These reactions were 50% inhibited by 7 microM KCN. The product of reduction of O2 catalyzed by the enzyme was concluded to be H2O on the basis of the ratio of ferrocytochrome c oxidized to O2 consumed.     

Cytochrome c oxidase of Pseudomonas AM 1: purification, and molecular and enzymatic properties

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Pseudomonas AM 1 to an electrophoretically homogeneous state and some of its properties were studied. The oxidase showed absorption peaks at 428 and 598 nm in the oxidized form, and at 442 and 604 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 602 nm. The enzyme molecule was composed of two kinds of subunits with molecular weights of 50,000 and 30,000 and it contained equimolar amounts of heme a and copper atom. The enzyme rapidly oxidized Candida krusei and horse ferrocytochromes c as well as Pseudomonas AM 1 ferrocytochrome c. The reactions catalyzed by the enzyme were strongly inhibited by KCN.     

Highly purified hydroxylamine oxidoreductase derived from Nitrosomonas europaea. Some physicochemical and enzymatic properties.

Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.     

Purification and characterization of membrane-bound CO-reactive hemoprotein from Tetrahymena pyriformis mitochondria

Abstract A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis . It showed absorption peaks at 615 and 455 nm in the reduced form and an α peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a ₆₂₀' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis , it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a ₆₂₀' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis .     

Cytochrome aa3 from the aerobic photoheterotroph Erythrobacter longus: purification, and enzymatic and molecular features

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Erythrobacter longus to homogeneity as judged by polyacrylamide gel electrophoresis, and some of its properties were studied. The spectral properties of the oxidase closely resembled those of mitochondrial and other bacterial cytochromes aa3. The enzyme showed absorption peaks at 430 and 598 nm in the oxidized form, and at 444 and 603 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 600 nm. The enzyme oxidized eukaryotic ferrocytochromes C more rapidly than E. longus ferrocytochrome c. The reactions catalyzed by the enzyme were 50% inhibited by 0.7 microM KCN. The enzyme contained 1 g atom of copper and 1 g atom of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two identical subunits, each with a molecular weight of 43,000.     

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.     

Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy

The b-type cytochrome in porcine neutrophils in situ was studied by the low temperature absorption spectroscopy at 77 K. Absolute spectra of the dithionite-reduced cell suspension revealed the existence of a b-type cytochrome with alpha, beta, and Soret absorption maxima at 558, 528, and 426 nm, respectively. The alpha band was unsymmetrical and showed a main peak at 558 nm with a shoulder at around 556 nm. When the cells were anaerobically stimulated either by phorbol myristate acetate or arachidonate followed by reduction by dithionite, the alpha band split clearly into double peaks at 555.5 and 558 nm, suggesting the presence of at least two states or species of the b-type cytochrome(s) in the cell. By monitoring absolute spectra of neutrophils at 77 K, we examined the possibility of CO binding to the b-type cytochrome. The absorption spectra of reduced b-type cytochrome in the presence and absence of CO, however, were not distinguishable under various conditions including equilibration with CO under high pressure or CO treatments in a dark room or at pH 8.5, 7.0, or 5.5. In contrast, the spectra of the reduced cytochrome disappeared immediately after exposure to O2, whether or not the cells had been treated with CO. The results indicate that the cytochrome does not form a CO complex in situ but reacts with O2, either directly or indirectly.     

Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties

Two isozymes of ascorbate (AsA) peroxidase were found in tealeaves, and one of them (AsA peroxidase II) was purified tohomogeneity, as judged by polyacrylamide gel electrophoresis.AsA peroxidase II is a monomer with a molecular weight of 34,000and contains protoheme, but it is not a glycoprotein. The enzymeshowed a Soret peak at 409 run and at 420 nm when oxidized andreduced, respectively, with an a-band at 556 nm. The oxidizedenzyme showed two small peaks at 478 nm and 530 nm. The peakat 478 nm disappeared when the enzyme was inactivated by depletionof AsA or by the addition of cyanide. Antibody raised againstAsA peroxidase II from tea did not cross-react with guaiacolperoxidase from spinach, and antibody against the guaiacol peroxidasedid not with AsA peroxidases from tea leaf. The amino acid compositionand amino acid sequence of the amino-terminal region of AsAperoxidase II were determined. Little homology in terms of aminoacid sequence was found between AsA peroxidase II and variousguaiacol peroxidases. The enzymatic and molecular propertiesof the two isozymes showed distinct differences with respectto molecular weight, sensitivity to AsA-depletion, specificityfor the electron donor, and other enzymatic properties. (Received April 13, 1989; Accepted July 25, 1989)     

Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.     

豆壳过氧化物酶的电子吸收光谱性质

应用电子吸收光谱技术研究了豆壳过氧化物酶 ( EC1 .1 1 .1 .7)的不同氧化态电子吸收光谱 ,并与其它来源的过氧化物酶作了比较研究 .天然态酶的特征吸收峰位为 40 4 nm的 Soret带 ,638nm的α带和 50 8nm的β带 ,与过氧化氢反应可生成三类复合物 .复合物 ( Com )在 40 8、580、61 8和 655nm处出现特征吸收 ;复合物 ( Com )在 41 9、52 9和 556nm处显示特征吸收 ;复合物 ( Com )则于 41 8、543和 578nm处显示特征吸收 .天然态酶经连二亚硫酸钠还原则出现 435和 558nm的特征峰 ,与氰化钾作用在 42 2和 544nm处显示特征吸收 .氰化钾对该酶的抑制为竞争性抑制 ,其 Ki 值为 2 .4μmol/L.     

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.     

Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R.

Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cytochrome a-type terminal oxidase derived from Thiobacillus novellus. Molecular and enzymatic properties

Cytochrome a-type terminal oxidase was purified from Thiobacillus novellus to an electrophoretically homogeneous state and some of its properties were studied.The enzyme shows absorption peaks at 428 and 602 nm in the oxidized form, and at 442 and 602 nm in the reduced form. The CO compound of the reduced enzyme shows peaks at 431 and 599 nm. The enzyme has 1 mol of haem a and 1 g-atom of copper per 55 600 g and is composed of two kinds of subunit, of 32 000 and 23 000 daltons, respectively.The enzyme reacts rapidly with tuna, bonito and yeast cytochromes c as well as with T. novellus cytochrome c, while it reacts slowly with horse and cow cytochromes c. The reduction product of oxygen catalysed by the enzyme is water.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

A CO-Binding Hemoprotein Derived from Tetrahymena pyriformis

A CO-binding hemoprotein was purified from Tetrahymena pyriformis and some of its properties were studied.

The hemoprotein possessed protoheme, its molecular weight was about 11,000, and its isoelectric point was at pH 8.2. The oxidized form of the hemoprotein showed the Soret band at 406 nm and had no distinct peaks in the region of α- and β-bands, while the reduced form showed the peaks at 426, 527 and 560 nm. The hemoprotein reacted with CO resulting in shift of the Soret band from 426 to 420 nm. The CO-compound showed a broad band from 537 to 573 nm. The hemoprotein was not autoxidizable or oxygenated either. It did not show either of the cytochrome oxidase, peroxidase and NADH oxidase activities.

It should be carefully determined whether or not cytochrome o is functioning as the terminal oxidase in T. pyriformis, as the CO-binding hemoprotein which does not react with molecular oxygen exists in the organism.     

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.