Oxidase Type Research Articles

Kinetics and mechanism for the binding of HCN to cytochrome c oxidase

The kinetics of cyanide binding to cytochrome c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k'obs) follows saturation type kinetics to form an HCN complex that subsequently undergoes a slow reaction (k'obs). The fast k'obs reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pKa = 7.45), while the other is that of HCN (pKHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k'obs reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k'obs reaction, the oxidase-HCN complex, still undergoes the slow k" process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k"obs) is independent of all the variations done in this study, and it probably corresponds to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monomeric or dimeric oxidase only slightly affects the magnitude of k'obs and k"obs values, and both processes are clearly present in both types of oxidase.

The highly conserved methionine of subunit I of the heme-copper oxidases is not at the heme-copper dinuclear center: Mutagenesis of M110 in subunit I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli

A common feature within the heme-copper oxidase superfamily is the dinuclear heme-copper center. Analysis via extended X-ray absorption fine structure (EXAFS) has led to the proposal that sulfur may be bound to CU B, a component of the dinuclear center, and a highly conserved methionine (M110 in the E. coli oxidase) in subunit I has been proposed as the ligand. Recent models of subunit I, however, suggest that this residue is unlikely to be near CU B, but is predicted to be near the low spin heme component of the heme-copper oxidases. In this paper, the role of M110 is examined by spectroscopic analyses of site-directed mutants of the bo 3-type oxidase from Escherichia coli. The results show that M110 is a non-essential residue and suggest that it is probably not near the heme-copper dinuclear center.

Three-dimensional crystals of cytochrome- c oxidase from Thermus thermophilus diffracting to 3.8 Å resolution

The ba 3-type cytochrome- c oxidase from Thermus thermophilus has been crystallized in its native form. Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl-β- d-thioglucoside as detergent. The crystals diffract to 3.8 Å, belong to the space group P2 or P2 1 and have unit cell dimensions of a = 80.7 A ̊ ; b = 116.0 A ̊ ; c = 156.9 A ̊ and β = 104.4°. The asymmetric unit contains two ba 3-type oxidase molecules.

The Cu Asite of the caa3-type oxidase of Bacillus subtilis is a mixed-valence binuclear copper centre

A copper-containing domain of the caa 3-type oxidase from Bacillus subtilis has been expressed as a water-soluble protein in the cytoplasm of Escherichia coli. Electron paramagnetic resonance (EPR) spectra of this purple domain show well-resolved lines in the g z resonance, both at X-band and S-band frequencies. Interpretation of EPR spectra and analytical data indicate a binuclear copper site consisting of one Cu 2+ and one Cu 1+. This copper site closely resembles Cu A in subunit II of cytochrome c oxidase and is shown here to be a mixed-valence [Cu 2+-Cu 1+) binuclear centre.

Kinetics of CO binding to H +-motive oxidases of the caa3-type from Bacillus FTU and of the o-type from Escherichia coli

The kinetics of CO rebinding with isolated Bacillus FTU caa 3-type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o-type oxidase as the main O 2reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO-oxidase complexes. The spectra of the optical absorbance changes upon photolysis were characteristic of CO- caa 3 and CO- o-oxidase complexes in Bac. FTU and E.Coli, respectively. Small quantities of d-type oxidase in E.Coli GO103 membranes were detected. The kinetics of CO reassociation with reduced caa 3 and o-type oxidases were monophasic with τ 25–30 ms in both cases.

Two types of terminal oxidase in alkalotolerant Bacillus FTU

The terminal segment of the Bacillus FTU respiratory chain has been studied. The shape of difference spectra and biphasic cyanide titration curve point to the presence of two terminal oxidases ( aa 3- and o-type) in the subcellular membrane vesicles of this Bacillus. The aa 3-type oxidase was shown to dominate in the beginning of the logarithmic phase while the o-type oxidase was more pronounced at the stationary phase of bacterial growth. Both enzymes oxidized TMPD in the presence of ascorbate: the o-type oxidase with K m = 0.7 mM TMPD and V max = 2.2 μmol O 2 min −1 nmol −1 haem o and the aa 3-type oxidase with K m = 2 mM TMPD and V max = 11.2 μmol O 2 min −1 nmol −1 haem a. KCN inhibited the two oxidases (half-maximal inhibition at 2 μM and 20 μM for the aa 3- and o-type oxidases, respectively).

Oxidation of methylamine by a Paracoccus denitrificans mutant impaired in the synthesis of the bc1 complex and the aa3-type oxidase Evidence for the existence of an alternative cytochrome c oxidase in this bacterium

A Paracoccus denitrificans fbcC—ctaDII double mutant strain impaired in the synthesis of both the bc 1 complex and the aa 3-type oxidase has been constructed. This mutant strain, which is still able to grown on methylamine as sole carbon and energy source, exhibits unimpaired oxygen consumption with succinate, methylamine and endogenous substrates as electron donors. From kinetic studies of the oxidation and reduction rates of cytochromes c, it can be concluded that P. denitrificans contains a second cytochrome c oxidase, different from the aa 3-type.

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.

Cytochrome oxidase in Anacystis nidulans: stoichiometries and possible functions in the cytoplasmic and thylakoid membranes

Three biochemically distinct membrane fractions were obtained from the cyanobacterium Anacystsis nidulans grown under salt-stressed conditions (0.4 M NaCl). These were classed as ICM (thylakoid), CM-1 (light cell membrane) and CM-II (heavy cell membrane). All three fractions were tested for cytochrome c oxidase activity. Two of the three, ICM and CM-II, exhibit such activity towards horse heart cytochrome c in both spectrophotometric and polarographic assay systems. In these two fractions, the oxidase, immunologically identical with other cytochrome aa 3 oxidases, was present at a much lower concentration than that of other cytochromes. ICM contain cytochrome b 6 f approximately equimolar with the photosystems (0.25μmol (g protein) −1 ); [cyt. aa 3 ] is between 2 and 5% [ b 6 f ]. CM-I has 1.5 μmol c -type cytochrome (g protein) −1 and a small amount of b -type cytochrome; [cyt. aa 3 ] is c ]. CM-II has 2.0 μmol c -type cytochrome (g protein) −1 and some b -type including a small b 6 f component; its [cyt. aa 3 ] is between 1 and 2% [cyt c ]. ICM oxidize cyanobacterial cytochrome c -554 ( c 6 ) in a reaction with the kinetic characteristics of an aa 3 -type oxidase. The donor for CM-II aa 3 also appears to be a (periplasmic) cytochrome c 6 . Small amounts of cytochrome aa 3 in ICM may be associated with a need to control the level of available reductant in a cyclic pathway. Slow reductant-level control could also be required in the heavy plasma membrane fraction (CM-II); the functions of extra c -type cytochromes in this membrane and in the oxidase-free CM-I fraction are at present unknown.

Bacillus subtilis chytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3‐type oxidases

The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127 degrees on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N',N'-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from ctaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify subunit II, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.

An alternative non-cytochrome containing branch in the respiratory system of free-living Rhizobium phaseoli

The existence of a NADH oxidase catalyzed by a non-cytochrome containing pathway in membranes of free-living Rhizobium phaseoli was explored. This alternative electron transport route was distinguished from the cytochrome-oxidase linked pathway by its low affinity towards O2 (K\(\left( {K_{m_{app} } = 75 - 80{\text{ }}\mu M} \right)\), higher Km for NADH (75 μM), a hundred-fold lower sensitivity to quinarine inhibition, and resistance to UV (360 nm) photoinactivation. In addition to NADH, tetramethyl-p-phenylenediamine (TMPD) donates electrons to this low-O2 affinity pathway, causing reduction bleaching of a flavoprotein absorption band at 455 nm. Ascorbate-TMPD dependent respiration was partially (25%) inhibited by 200 μM quinacrine. The low O2-affinity oxidase activity promoted by NADH, or ascorbate plus TMPD was present in aerobic and microaerophilic grown cells and absent in anaerobic and bacteroid cells. Thus, a NADH linked flavoprotein type oxidase is suggested.

The o-type oxidase of the acidophilic methylotroph Acetobacter methanolicus

Membranes of the acidophilic methylotroph Acetobacter methanolicus contained only b- and c-type cytochrome and a CO-binding b-type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome c and ascorbate/TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent3-12 (1.7 fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (200 mM-NaCl) and stable between pH 4.0 and 6.8. Of the two soluble c-type cytochromes from A. methanolicus only the typical class I cytochrome (cytochrome c H) was a good substrate, as was equine cytochrome c. The oxidase was partially purified by anion-exchange chromatography but further purification proved impossible. The yield with respect to equine cytochrome c oxidation was 18%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome c H and TMPD was lost. Neutral phospholipids had little effect on activity of the oxidase but the charged phospholipids phosphatidylglycerol and phosphatidylserine stimulated activity up to about fourfold. It proved impossible to incorporate the oxidase into active lipoprotein vesicles. During the purification process the pH optimum for oxidation of cytochrome c H was unchanged (pH 5.6) whereas that for oxidation of equine cytochrome changed from pH 9.5 to 7.5 and the sensitivity of the oxidase to azide changed from non-competitive to competitive during the purification process. The partially-purified oxidase contained only b-type cytochrome, some of which was CO-reactive. It is proposed that the oxidase is a cytochrome co type of oxidase that loses its cytochrome c component during the purification process and is only able to oxidize c-type cytochromes if these can be formed into a ‘reconstituted’ cytochrome co with the partially-purified oxidase.

Structure-modulated recognition of substrates and inhibitors by monoamine oxidases A and B

Introduction Two types of the flavin-containing monoamine oxidase (MAO, EC 1.4.3.4) termed MAO-A and MAO-B have been described on the basis of substrate specificity and drug sensitivity. The two forms are composed of one or two identical or slightly different FAD-bearing subunits of similar molecular mass (approx. 60 kDa) and appear to derive from separate genes [ 11. Both forms of MA0 are localized in the outer mitochondria1 membrane and the activity of MAO-A might be more sensitive to the phospholipid microenvironment than that of MAO-B [2]. Very little is known as to the molecular structure of the active site of the two isoenzymes, except that both forms of MA0 have identical sequences of amino acids immediately adjacent to the covalently bound flavin [Ser-Gly-Gly-Cys (FAD)-Tyr] [3]. Since it is not possible to obtain direct information on the catalytic site of MA0 in its native form, some insight into the topography of the active site might be acquired by using selective substrates and, better still, selective inhibitors of either form of MAO.

STUDIES ON MITOCHONDRIAL METABOLIC PROCESSES IN OFFSPRING OF ALCOHOLIZED RATS — I. EVIDENCE FOR ALTERED ACTIVITY AND SENSITIVITY TO MONOAMINE OXIDASE-DEPENDENT CONTROL BY BIOGENIC AMINES OF SOME MEMBRANE-BOUND ENZYMES

In the liver mitochondrial fraction of the first generation offspring of alcoholized male rats, decreased activities of monoamine oxidase (MAO) types A and B, rotenone-insensitive NADH-cytochrome c-reductase and succinate dehydrogenase were observed. The MAO-dependent inhibition of rotenone-insensitive NADH-cytochrome c-reductase and succinate dehydrogenase by biogenic amines, incubated with the mitochondrial fraction, was altered in the offspring of alcoholized animals as compared with control rats. The sensitivity of these enzymatic activities towards the inhibitory effect of 5-methoxyindol-3-ylacetaldehyde was markedly increased in the offspring of alcoholized male rats. The data obtained suggest the existence of a genetically determined predisposition of the mitochondrial metabolic processes in the offspring of the alcoholized rats to the effects of ethanol and to the toxic effects of acetaldehyde, formed during ethanol metabolism.

Effect of site-specific mutagenesis of tyrosine-55, methionine-110 and histidine-217 in porcine kidney D-amino acid oxidase on its catalytic function

To assess the contributions of Tyr-55, Met-110 and His-217 in porcine kidney D-amino acid oxidase (EC 1.4.3.3, DAO) to its catalytic function, we constructed three mutant cDNAs coding for the enzymes possessing Phe-55, Leu-110 and Leu-217 by site-specific mutagenesis. The mutant and wild type cDNAs could be expressed in vitro with similar efficiency. The three mutant enzymes thus synthesized showed catalytic activities comparable to that of the wild type oxidase. It is concluded that Tyr-55, Met-110 and His-217 are not directly involved in the catalytic function.

Platelet monoamine oxidase B: use and misuse.

The human platelet in addition to having serotonin (5-HT) receptors, uptake carriers (receptor) and transmitter storage vesicles, primarily possesses mitochondrial monoamine oxidase (MAO) type B. Similar to the major form of MAO in the human brain, this enzyme actively oxidizes A-B and B substrates (tyramine, dopamine, phenylethylamine) as well as the novel secondary amine anticonvulsant, milacemide and dopaminergic neurotoxin, MPTP. 5-HT oxidation is hardly affected by the platelet enzyme and MAO inhibitors have no net effect on its accumulation. MAO-B is selectively inhibited by 1-deprenyl and thus the platelet enzyme may be useful to monitor the anti-Parkinson activity of such drugs, as related to their ability to inhibit brain MAO-B. The oxidation of the anticonvulsant, milacemide, to glycine in vitro and in vivo by MAO-B, may herald new prospects for the development of inert prodrugs capable of being metabolized to neuroactive substances by MAO-B. The plasma levels of their metabolites may be an index of MAO-B activity found in the platelet and brain.

Inhibition of 5-hydroxytryptamine accumulation and deamination by substituted phenylalkylamines in hypothalamic synaptosomes from normal and reserpine-pretreated rats

1. In the present study the abilities of different compounds to inhibit MAO inside and outside the serotonergic neurons, to inhibit the accumulation of 5-HT and to release 5-HT were separated by using different in vitro techniques. With these methods a number of substituted phenylalkylamines, which are reversible inhibitors of monoamine oxidase (MAO) type A, were characterized. 2. The compounds were examined regarding their ability to inhibit the accumulation of 5-HT and to inhibit MAO in the same synaptosomal preparation of hypothalamus from normal and reserpine-pretreated rats. The difference in the uptake of 14C-5-HT (0.1 mumol/l) in the absence and presence of citalopram (0.25 mumol/l) was taken as a measure of the accumulation into the serotonergic synaptosomes. The deamination of 14C-5-HT (0.1 mumol/l) in the presence of citalopram (0.25 mumol/l) was considered as that brought about outside the serotonergic synaptosomes, whereas the difference between the deamination in the absence and presence of citalopram was taken as the MAO activity inside the serotonergic synaptosomes. 3. Most of the phenylalkylamines were slightly more potent as MAO inhibitors outside serotonergic synaptosomes than as inhibitors of 5-HT accumulation in normal rats. The most potent MAO inhibitors, both in absolute terms and in comparison with uptake inhibitory potency, were the 2,6-dichloro-(FLA 365) and the phenylpropylene-(FLA 417) derivatives. 4. A difference in potency on the accumulation in synaptosomes from normal and reserpine-pretreated rats was found for many of the phenylalkylamines with the exception of FLA 365, FLA 417 and the 2,5-dimethyl derivative RAN 113.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxisomal oxidation of L‐2‐hydroxyphytanic acid in rat kidney cortex

A previously unreported metabolite of mammalian phytanic acid catabolism, 2-oxophytanic acid, was identified by gas chromatography/mass spectrometry analysis. The formation of 2-oxophytanic acid was demonstrated to result from the oxidation of L-2-hydroxyphytanic acid, a reaction catalysed by a rat-kidney-cortex H2O2-generating oxidase. The pH optimum for the L-2-hydroxyphytanate oxidase activity was 8.5 and its apparent Km and Vm were about 0.15 mM and 0.35 mumol min-1 (g tissue)-1, respectively. L-2-Hydroxyisocaproate, a substrate of rat kidney L-alpha-hydroxyacid oxidase type B, inhibited the formation of 2-oxophytanate from L-2-hydroxyphytanic acid. Fractionation studies have indicated that 40% of L-2-hydroxyphytanate oxidase was associated with a particulate fraction and that the activity distribution of the oxidase closely paralleled that of catalase, a well known peroxisomal marker enzyme.

Effect of carbon tetrachloride intoxication on the type conversion of xanthine dehydrogenase into xanthine oxidase in rats

The conversion of xanthine dehydrogenase(type D) into xanthine oxidase(type O) was significantly increased in serum and liver of all CCl4 treated rats on the necrosis and early cirrhosis stage of liver tissue. In the pretreatment of prednisolone, the ratio of type O per type O+D showed the decreasing tendency in serum but the significant decrease in liver.In vitro, the conversion of liver xanthine oxidase from type D into type O was markedly increased by following preincubation with lysosomal fraction. The type conversion of xanthine oxidase may be caused by proteolytic enzymes in lysosome.

Monoamine oxidase types A and B in the human adrenal gland and pheochromocytoma

The distribution of the two types of monoamine oxidase within the human adrenal gland was studied. As demonstrated by histochemical techniques, the zona reticularis of the adrenal cortex contains the type A enzyme and the zonas glomerulosa and fasciculata contain little enzyme activity. Biochemical analysis showed that the adrenal medulla contains both types of monoamine oxidase. It is suggested that the type B enzyme is present in chromaffin cells and the type A enzyme is present in vascular and other neural elements. Pheochromocytoma, a tumor of the adrenal medulla, apparently does not contain the B type enzyme.