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.     

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.     

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.     

Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans

Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly to homogeneity by an anaerobic procedure that results in a product with high specific activity. The enzyme is a dimer of about Mr 144,000 composed of two subunits of apparently equal Mr and contains 4 mol of Cu per mol of subunit. The isoelectric point is 4.3; specific activity at 25 degrees C, pH 7.1, is 122 mumol X min-1 X mg of protein-1; and Km is about 7 microM N2O under the same conditions. The N2O- and O2-oxidized forms of the enzyme had principal absorption bands at 550 and 820 nm; the dithionite-reduced form, at 650 nm. The extinction coefficient at 550 nm for the oxidized enzyme is about 5300 (M subunit)-1 X cm-1. Ferricyanide-oxidized enzyme and enzyme exposed to O2 for a couple of days at 4 degrees C exhibited additional bands at 480, 620, and 780 nm and had very low specific activities. Cu-EPR signals were observed with oxidized and reduced forms of the enzyme with g perpendicular values at 2.042 and 2.055, respectively. The O2-oxidized enzyme had g parallel and A parallel values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g parallel region. The enzyme may therefore contain at least one Cu atom approximating the "Type 1" class. Spin counts against Cu-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the O2-oxidized enzyme and 7-15% in the enzyme as prepared and in the reduced enzyme. Much of the Cu thus appears to be EPR silent. Nitrous oxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluoride among other anions were found to accelerate this process. In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfectomarina and Rhodopseudomonas sphaeroides, particularly the former. Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions.     

Purification and properties of a peroxidase from Halobacterium halobium L-33

A peroxidase was purified from Halobacterium halobium L-33 to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed an absorption peak at 406 nm in the oxidized form and peaks at 440, 558, and 591 nm in the reduced form. The difference spectrum, reduced + CO minus reduced, of the enzyme showed peaks at 425, 538, and 577 nm and troughs at 444, 562, and 596 nm. These spectral properties were apparently similar to those of "cytochrome a1" except for the occurrence of the peak at 558 nm in the reduced form. The molecular weight of the enzyme was 110,000 and the enzyme possessed one unit of protoheme in the molecule. The activity to oxidize guaiacol in the presence of H2O2 of the peroxidase was about one-twentieth of that of horseradish peroxidase. The enzyme also showed a catalase-activity one-fourth as active as that of liver catalase. The reactions catalyzed by the enzyme were strongly inhibited by KCN.     

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase.

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.     

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.     

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).     

Binding of ligands and spectral shifts in cytochrome c oxidase

1. On addition of reductant (ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa(3)), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415nm, 445nm and 605nm, which slowly gives rise to the final ;half-reduced' species in whose spectrum the 415nm peak has disappeared and a new absorption is seen at 430-435nm. 2. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the ;low-spin' cyanide form with a peak at 432nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally ;mixed-spin', undergoes no further changes. 3. The Soret-peak shift of oxidized cytochrome a(3) which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605nm to 603nm. A partial blue shift of the alpha-peak occurs in the half-reduced sulphide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulphides [a(2+)a(3) (3+)HSR compounds, where R=CH(3), C(2)H(5) or (CH(3))(2)CH]. 4. Analogous, albeit less readily decipherable, spectroscopic effects with the ligands imidazole and alkyl isocyanides suggest that on reduction of cytochrome a an interaction occurs between the two haem groups involving (i) a high- to low-spin change in cytochrome a(3), and after this, (ii) a change in the molecular environment of the cytochrome a. The latter effect, possibly a decrease in the hydrophobicity of the haem pocket, requires that the ligands on cytochrome a(3) have a bulky and partially hydrophobic character.     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

Effect of pH on the spectrum of cytochrome c oxidase hydrogen peroxide complex

Hydrogen peroxide binding to ferric cytochrome c oxidase in proteoliposomes brings about a red-shift of the enzyme Soret band and increased absorption in the visible range with two prominent peaks at approx. 570 and 607 nm. The molar absorptivity of the H2O2-induced difference spectrum is virtually pH-independent in the Soret band and at 570 nm, whereas the peak at 607 nm increases approx. 3-fold upon alkalinization in a narrow pH range 6.0-7.2, the effect being reversible. The pH profile of this transition indicates ionization of two acid-base groups with close pK values of 6.7. The lineshape of the peroxide compound difference spectrum is found to respond to pH changes inside the proteoliposomes. It is suggested that peroxide-complexed enzyme can undergo a pH-dependent transition to a form with increased extinction at 605-607 nm, possibly corresponding to the 420 nm (or 'pulsed') conformer of the ferric cytochrome oxidase formed as an early product of the enzyme oxidation. Accordingly, relaxation of the '420 nm' form to the resting state would be linked to an uptake of two protons from the M-aqueous phase. This protolytic reaction might be a partial step of the cytochrome oxidase proton pumping mechanism or it could serve to regulate interconversion between the active 'pulsed' and less active 'resting' states of the enzyme in the membrane.     

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

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 a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

Naphthalene dioxygenase: purification and properties of a terminal oxygenase component

Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a three-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, oxygen, and partially purified preparations of components A and C. An estimated Mr of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of two subunits with molecular weights of ca. 55,000 and 20,000, indicative of an alpha 2 beta 2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462, and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other two components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH, and O2. These results, together with the detection of the presence of 6.0 g-atoms of iron and 4.0 g-atoms of acid-labile sulfur per mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an iron-sulfur protein which functions in the terminal step of naphthalene oxidation.     

Phospholipid oxidation catalyzed by cytochrome c in liposomes

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.     

Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

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).