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.     

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.     

Thiobacillus novellus cytochrome c oxidase contains one heme alpha molecule and one copper atom per catalytic unit.

The minimal structural unit of cytochrome c oxidase purified from Thiobacillus novellus was composed of one molecule each of two subunits with molecular masses of 32 and 23 kDa, respectively, and the unit had one molecule of heme a and one atom of copper. In the presence of n-octyl-beta-D-thioglucoside, the oxidase existed as the monomeric form of the unit, while it occurred as the dimeric form of the unit in the presence of Tween 20. The monomeric form showed an active cytochrome c oxidizing activity and reduced molecular oxygen to water with ferrocytochrome c. Namely, it has been shown that the bacterial cytochrome c oxidase with one heme a molecule and one copper atom per molecule can catalyze oxidation of ferrocytochrome c with concomitant reduction of molecular oxygen to water.     

A relationship between prokaryote and eukaryote observed in Nitrobacter agilis cytochromes AA3 and C

Cytochrome aa3 (cytochrome c oxidase) and cytochrome c were purified from Nitrobacter agilis, and some of their properties were compared with those of the respective counterparts of eukaryote from the evolutionary point of view. N. agilis cytochrome aa3 has many functional and structural properties similar to those of eukaryotic cytochrome aa3, although its molecule is composed of only two kinds of subunits unlike the eukaryotic cytochrome which is composed of 7 kinds of subunits. N. agilis cytochrome c is homologous to eukaryotic cytochrome c; 50 amino acid residues of the bacterial cytochrome c are identical with those of horse cytochrome c. It reacts with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochrome c does. So far as based on the molecular features of cytochromes aa3 and c, N. agilis appears to be one of the organisms which may link in evolution prokaryote to eukaryote.     

A comparison between Nitrosomas europaea and Thiobacillus novellus on the basis of their oxidation systems of inorganic compounds.

Nitrosomonas europaea and Thiobacillus novellus were compared with each other on the basis of the biochemical properties of their inorganic compound-oxidizing systems. Cytochromes c of the two organisms differ considerably from each other; N. europaea cytochrome c-552 belongs to the "bacterial-type" cytochrome c, while T. nouellus cytochrome c-550 resembles eucaryolic cytochrome c. The specificity of cytochrome oxidase for cytochrome c as the electron donor is different between the two organisms; T novellus oxidase reacts rapidly with cytochromes c of the organisms which seem to be higher than the organisms whose cytochromes c react rapidly with N. europaea oxidase. On the basis of these facts, N. europaea seems to be older organism than T. novellus in terms of evolution.     

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.     

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.     

A novel aco-type cytochrome-c oxidase from a facultative alkalophilic Bacillus: purification, and some molecular and enzymatic features.

A novel aco-type cytochrome-c oxidase was highly purified from the facultative alkalophilic bacterium, Bacillus YN-2000, grown at pH 10. The enzyme contained 9.0 nmol heme a/mg protein. It contained 1.23 mol of protoheme, 1.06 mol of heme c, 2.0 g atoms of copper, 2.5 g atoms of iron, and 1.8 g atoms of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two subunits with Mrs of 52,000 and 41,600. On the basis of these results, the enzyme seemed to contain one molecule each of heme a, protoheme, and heme c per minimal structural unit (Mr, 93,600). Only protoheme among the three kinds of hemes in the enzyme reacted with CO and CN-. Heme a did not react with CO; cytochrome a3 did not seem to be present in the enzyme. The enzyme oxidized 314 mol of horse ferrocytochrome c per heme a per sec at pH 6.5 and the catalytic activity was 50% inhibited by 7.65 microM KCN. The enzymatic activity was found to be optimal at pH 6.0.     

Methylobacillus flagellatus KT contains a novel cbo-type cytochrome oxidase

The o-type oxidase from the methanol-grown obligate methylotroph Methylobacillus flagellatus KT has been purified to homogeneity. The complex is composed of four subunits (57, 40, 35 and 30 kDa). It contains six haems (4C:1B:1O) and one copper atom per molecule. It is proposed that the haem O-Cu(B) binuclear centre and a low-spin haem B are located in subunit I (57 kDa), two haems C reside in the cytochrome c homodimer (35 kDa), two haems C belong to the dihaem cytochrome c (30 kDa). The presented data provide evidence that cytochrome cbo is a novel representative of the haem-copper oxidase superfamily.     

Cytochrome aa3 from Nitrosomonas europaea

Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.     

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.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a1c1 is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c-550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a1c1 and aa3-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c-550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP+ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi. The electron transfer against redox potential from NO2- to cytochrome c could be pushed through prompt removal by cytochrome aa3 of H+ formed by the dehydrogenation of NO2- + H2O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO2-, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a1c1 with NO2- in vivo.     

The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase. ATP affects the oxidase uniquely.

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady-state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]-v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis-Menten-type hyperbola in the presence of 700 microM ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 microM ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 microM ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]-v curve and the molecular mass of the oxidase when used in place of ATP.     

Some properties of Nitrosomonas europaea cytochrome c oxidase (aa3-type) which lacks CuA

From Nitrosomonas europaea which had been cultivated in a medium deficient in copper, cytochrome c oxidase (aa3-type) which did not have CuA was purified. The oxidase did not show the 830-nm peak and its ESR spectrum differed greatly from that of the normal enzyme, which has two copper atoms, CuA and CuB, per molecule. However, the oxidase which did not have CuA showed almost the same cytochrome c oxidizing activity as the normal oxidase.     

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)     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

The photoreactivity of the copper-NO complexes in cytochrome c oxidase and in other copper-containing proteins

The complexes of NO with CuB of cytochrome c oxidase in which cytochrome a3 may or may not be ligated to cyanide or fluoride are photodissociable. NO does not appear to react with CuB in complexes of cytochrome c oxidase in which sulphide or mercaptans are ligated to the haem iron of cytochrome a3. A comparison is made between the photoreactivity of the complexes of NO with cytochrome c oxidase and those with ceruloplasmin, ascorbate oxidase, and haemocyanin. It is shown that the photoreactivity of CuB 2+.NO in cytochrome c oxidase is not unique for this enzyme, but may also be observed in the complexes of NO with type-1 copper-containing enzymes. This would suggest that the ligation of CuB in cytochrome c oxidase shows some similarity to type-1 copper in blue oxidases.     

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.     

Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits.

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.     

A cytochrome o-type oxidase of the thermophilic bacterium PS3 grown under air-limited conditions

A cytochrome o-type oxidase from the thermophilic bacterium PS3 grown under air-limited conditions was purified by ion-exchange chromatography in the presence of a non-ionic detergent. The enzyme was composed of three subunits (60, 30, and 16 kDa) and seemed to contain two molecules of heme b as prosthetic groups. It contained no detectable copper. The reduced enzyme showed absorption bands at 426 and 558.5 nm, and a characteristic spectral change upon binding CO. It oxidized several cytochromes c and artificial dyes such as N,N,N',N'-tetramethyl-p-phenylenediamine and phenazonium methosulfate at appreciable rates. Its Km for O2 was low (0.09 microM). It was capable of transmembrane electron transfer, because when reconstituted into liposomes, it generated a membrane potential upon oxidation without pumping protons.