Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

A comparison of the local environment of Cu(II) in native and half-Cu-depleted bovine serum amine oxidase

Electron spin-echo envelope modulation spectroscopy has been used to compare the local environment of Cu(II) in native bovine serum amine oxidase, containing two copper atoms/dimer molecule, and in a protein preparation, half depleted of copper, which has little enzymatic activity. For each preparation, two different populations of coordinated imidazoles with inequivalent magnetic coupling to copper could be recognized. In addition, water was shown to be a ligand to copper. No differences in coordinated ligand structures between the native and half-Cu-depleted preparations could be seen. In addition, the amount of ambient, non-coordinated water detected for native and half-Cu-depleted proteins was found to be nearly equivalent. However, the addition of phenylhydrazine, an inhibitor that binds to the pyrroloquinoline quinone cofactor but not to Cu(II) in the native enzyme, displaces ambient water near copper.     

Investigation of Cu(I)-dependent 2,4,5-trihydroxyphenylalanine quinone biogenesis in Hansenula polymorpha amine oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.     

Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.     

Studies on sulfhydryl groups of Aspergillus niger amine oxidase.

Amino acid analysis of the amine oxidase of Aspergillus niger (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) showed a composition similar to that of bovine plasma enzyme. One molecule of enzyme contained 25 Cys residues. It was shown that 9 to 11 residues of Cys were titrated to be SH groups. The amine oxidase reaction was markedly inhibited by metal ions (Cu2+, Hg2+, Ag+). The enzyme was inactivated with SH reagents (phenyl mercuric acetate, Cl-HgBzO-) and the extent of this inactivation was dependent on the time of incubation with SH reagents. Also, the Cl-HgBzO- -inactivated enzyme was reactivated with cysteine and this reactivation was biphasic with the time of incubation. The Cl-HgBzO--inactivated amine oxidase was compared with the native enzyme in their reactivity with phenylhydrazine and their spectral properties. The results showed that the Cl-HgBzO--inactivated enzyme had lower reactivity with phenylhydrazine than the native enzyme and had higher absorbance values than the native enzyme around 400 nm wavelengths.     

Removal of type 2 Cu from ascorbate oxidase and laccase by reaction with n,n-diethyldithiocarbamate

The type 2 Cu of ascorbate oxidase from zucchini peelings can be rapidly removed by reaction with a tenfold excess N,N-diethyldithiocarbamate (DDC) in air, while other chelating agents, such as EDTA, require anaerobic reducing conditions. The type 2 Cu of laccase from Rhus vernicifera is never removed under aerobic conditions. In anaerobiosis and in the presence of a reducing agent, EDTA is also unable to remove the copper unless a smaller lipophilic molecule (DDC or dimethylglyoxime) is present, acting as a mediator. Type 1 Cu is not involved in the reaction of ascorbate oxidase with DDC, but reduction of type 3 Cu is probably required for type 2 Cu depletion, suggesting interdependence of type 2 and type 3 copper. Type 2 Cu is less exposed in laccase, possibly because of the large carbohydrate content of this protein.     

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.     

Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate

The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM.     

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.     

Cryoenzymology and spectrophotometry of pea seedling diamine oxidase

Diamine oxidase follows bi-ter ping-pong kinetics, with an intermediate, "reduced" free-enzyme form being generated after the anaerobic conversion of amine to aldehyde. Visible spectra of diamine oxidase reacting at subzero temperatures provide evidence that this intermediate enzyme form is obtained via several other intermediates and that the environment of the Cu(II) changes dramatically during the course of the reaction [even though it is not reduced to Cu(I) during the catalytic cycle]. The spectrum of this form of diamine oxidase, which is obtained 0.5--2 h after the addition of amine at -5 to -15 degrees C, is independent of substrate, is identical with that obtained by anaerobic addition of substrate at room temperature, and provides evidence for a direct interaction of Cu(II) with the organic cofactor of the enzyme. This interaction is apparently charge transfer in nature. Upon removal of Cu(II) from the native enzyme, one obtains spectral evidence that the organic cofactor is still present. However, removal of the Cu(II) from the reduced (intermediate) enzyme form yields a featureless enzyme spectrum and a Cu(II)--chelate complex which contains a new ligand, which is presumably the second prosthetic group.     

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5''-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.     

Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses

Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.     

Characterization of a model compound for the lysine tyrosylquinone cofactor of lysyl oxidase

We characterized a model compound for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase which is one of the mammalian copper-dependent amine oxidases. The model compound, 4-butylamino-5-methyl-o-quinone, was prepared from n-butylamine and 4-methylcatechol by the oxidation with sodium iodate and characterized by spectroscopic analyses. The absorption maximum at 494 nm is consistent with that of lysyl oxidase. The model compound was capable of deaminating benzylamine to benzaldehyde at 37 degrees C in buffered aqueous acetonitrile. The aldehyde production was markedly elevated in the presence of the Cu(II)-EDTA complex but inhibited by free Cu(II). The catalytic cycle was observed at pH 10 in the presence of Cu(II), and the pH activity profile showed a broad optimum at about pH 9.0. In the presence of beta-aminopropionitrile and upon deoxygenation with N2 aldelyde, production was decreased. The important features of the reaction were consistent with the enzymatic reaction.     

Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: modulation by anaerobiosis, certain antioxidants and oxidants.

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

The octapeptide repeat region of prion protein binds Cu(II) in the redox-inactive state

The octapeptide repeat region of human prion protein is known to bind four Cu(II) ions per molecule. A peptide, Octa(4), representing this region was tested for inhibitory effects on copper-catalyzed oxidation of l-ascorbate or glutathione and on generation of OH(*) during the former reaction. The result indicated that the catalytic activity of the first Cu(II) ion bound to an Octa(4) molecule was completely suppressed. The valence state of the copper under reducing conditions was Cu(II), as determined by a newly developed method using bathocuproinedisulfonate under acidic conditions. Furthermore, it was shown that Escherichia coli cells expressing the octapeptide repeat region were significantly resistant to copper treatment compared with control cells. The results taken together indicate that prion protein can function to sequester copper ions in the redox-inactive state, rendering copper-induced generation of reactive oxygen species impossible.     

Proton relaxation study of the hog kidney diamine oxidase active center.

Proton relaxation studies of the interactions with hog kidney diamine oxidase of water, substrate-analogue inhibitors, and product analogues indicate that the active site Cu(II) is not located near the oxidizing site of the enzyme, rather near the nonoxidized end of the binding substrate. The studies with histamine derivatives provide evidence for a concentration-dependent occupation of two sites. The site which is populated at high concentrations provides proximity of the imadazole ring nitrogen N1 to the Cu(II). Water binds at the Cu(II) of the native enzyme. However, this water is probably not involved in the hydrolysis of the enzyme-substrate imine bond to eliminate the first reaction product. O2 does not compete with H2O for a site on the Cu(II) ion. In the case of one of the probes, namely the ammonia (product) analogue dimethylamine, the validity of the protein relaxation results was verified by also observing the nitrogen (15N) relaxation rates of ammonia itself. The conclusion that the ammonium ions is not directly bonded to the active site Cu(II) is reached from both the proton and nitrogen relaxation experiments.     

Spectroscopic observation of intermediates formed during the oxidative half-reaction of copper/topa quinone-containing phenylethylamine oxidase.

The catalytic reaction of copper/topa quinone (TPQ) containing amine oxidase consists of the initial, well-characterized, reductive half-reaction and the following, less studied, oxidative half-reaction. We have analyzed the oxidative half-reaction catalyzed by phenylethylamine oxidase from Arthrobacter globiformis (AGAO) by rapid-scan stopped-flow measurements. Upon addition of dioxygen to the substrate-reduced AGAO at pH 8.2, the absorption bands derived from the semiquinone (TPQ(sq)) and aminoresorcinol forms of the TPQ cofactor disappeared within the dead time (<1 ms) of the measurements, indicating that the reaction of the substrate-reduced enzyme with dioxygen is very rapid. Concomitantly, an early intermediate exhibiting an absorption band at about 410 nm was formed, which then decayed with a rate constant of 390 +/- 50 s(-1). This intermediate was detected more prominently in the reaction in D2O buffer (pD 8.1) and was assigned to a Cu(II)-peroxy species. The assignment was based on the observation that addition of H2O2 to the substrate-reduced AGAO under anaerobic conditions led to the formation of a new band at about 415 nm, accompanied by partial quenching of absorption bands derived from TPQ(sq). Other intermediates exhibiting absorption bands at about 310 and 340 nm were also observed in the oxidative half-reaction. Kinetics of the disappearance of these latter bands did not correspond with that of the Cu(II)-peroxy band at 410 nm but did well with that of the increase of the 480 nm absorption band due to the reoxidized TPQ. Rapid increase of the absorption in the 320-370 nm region was also observed for the reaction of the substrate-reduced, Ni-substituted enzyme with dioxygen. On the basis of these results, a possible mechanism is proposed for the oxidative half-reaction of the bacterial copper amine oxidase.     

Properties of copper-free pig kidney amine oxidase: role of topa quinone

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.