Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.     

Speciation of phytate ion in aqueous solution. Protonation constants and copper(II) interactions in NaNO3aq at different ionic strengths

The acid base behavior of phytate has been studied (at t=25 degrees C by potentiometry, ISE-H+ glass electrode) in NaNO3aq at different ionic strengths (0.1 < or = I/mol L(-1) < or = 1.0). The interactions with copper(II) were investigated in the same experimental conditions in different metal to ligand (Phy) ratios (1:1 < or = Cu2+ :Phy < or = 4:1), by using both ISE-H+ and ISE-Cu2+ electrodes. Phytate acid base behavior in sodium nitrate is very similar to that in sodium chloride, previously investigated. In the experimental conditions adopted, the formation of three CuiHjPhy(12-2i-j)- species is observed: the mononuclear CuH4Phy6- and CuH5Phy5-, and the dinuclear Cu2H5Phy3-. Analysis of complex formation constants at different ionic strengths reveals that both ISE-H+ and ISE-Cu2+ electrodes gave, within the experimental error, analogous values. Dependence of complex formation constants on ionic strength was modeled by EDH (Extended Debye-Hückel) and SIT (Specific ion Interaction Theory) equations. The sequestering ability of phytate toward copper(II) has been evaluated by the calculation of pL50 (the total ligand concentration, as -log CL, able to bind 50% of metal cation), an empirical parameter already proposed for an objective "quantification" of this ability. A thorough analysis of literature data on phytate-copper(II) complexes has been performed.     

In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus

The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.     

Some aspects of the copper(II)-DNA interaction

The Cu(II) ion interaction with calf-thymus DNA was studied by means of differential pulse polarography and sweep voltammetry as well as chromatography and viscosimetry. Most of the complexes formed at high ionic strength (0.2 M) and lower Cu(II) concentrations are of a nondenaturing nature. Their formation has but a minor effect on unwinding process of the DNA double helix. The excess of Cu(II) (P = 5) leads, however, to distinct denaturation of the DNA structure. Metal ions have little effect on the denaturation induced by the polarographic reduction of DNA on the mercury electrode. This conclusion is consistent with the character of the polarographic process and with the fact that Cu(II) ions are not very effective in the interaction with AT pairs. Cupric ions have no renaturing ability towards thermally denatured DNA at 0.2 M ionic strength but distinct renaturation was observed at low ionic strength (0.05 M).     

Soluble CuA domain of cyanobacterial cytochrome c oxidase

The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa(3)-type cytochrome c oxidase. The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes. Here we report the expression and purification of a truncated subunit II copper A (Cu(A)) domain (i.e. the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield. The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV. Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa(3)-type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu(A) domains. Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics. At pH 7 and low ionic strength the bimolecular rate constant is (2.1 +/- 0.3) x 10(4) m-1 s(-1), and the rates decrease upon the increase of ionic strength. Sequence alignment and modeling of cyanobacterial Cu(A) domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu(A) domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR. The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs. NHE at pH 7.0, respectively. Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.     

The reaction sequence of the Na+/K(+)-ATPase: rapid kinetic measurements distinguish between alternative schemes.

Conformational changes between E1 and E2 enzyme forms of a dog kidney Na+/K(+)-ATPase preparation labeled with 5-iodoacetamidofluorescein were followed with a stopped-flow fluorimeter, in terms of the rate constant, kobs, and the steady-state magnitude, % delta F of fluorescence change. On rapid mixing of enzyme plus Mg2+ plus Na+ with saturating (0.5 mM) ATP in the absence of K+, kobs varied with Na+ concentration in the range 0-155 mM, with a K1/2 of 10 mM, while % delta F was relatively insensitive to Na+, with a K1/2 of 0.5 mM. Oligomycin reduced kobs by 98-99% for Na+ greater than or equal to 10 mM, but only by 50% for Na+ = 1 mM; % delta F was reduced at most by 20%. At 155 mM Na+, both kobs and % delta F changed if K+ was present with the enzyme. kobs decreased by 50% when K+ was increased from 0 to 0.2 mM, but increased when K+ was varied in the range 0.2-5 mM. K+ increased % delta F by a factor of 3 with a K1/2 of 0.3-0.5 mM as measured in both stopped-flow and steady-state experiments. These data are considered in terms of the derived presteady-state equations for two alternate schemes for the enzyme, with the E1P to E2P conformational change either preceding (Albers-Post) or following (N?rby-Yoda-Skou) Na+ transport and release. The analysis indicates that: (i) Na+ must be released before the conformational transition, from an E1 form; (ii) the step in which the second and/or third Na+ is released is rate-limiting, but this release is accelerated by Na+; and (iii) the release is also accelerated by K+ acting with low affinity (possibly at extracellular sites).     

113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.     

X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase

X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state shows a peroxide bridging between Fe2+ and Cu2+ in the O2 reduction site. The bond distances for Fe-O and Cu-O are 2.52 and 2.16 A, respectively. The structure is consistent with antiferromagnetic coupling between the two metals, which has long been known and to recent redox titration results [J. Biol. Chem. 274 (1999) 33403]. The trigonal planer coordination of Cu1+ in the O2 reduction site is consistent with the very weak interaction between Cu1+ and O2 bound at Fe2+ revealed by time-resolved resonance Raman investigations. One of the three histidine imidazoles coordinated to the Cu ion in the O2 reduction site fixes a tyrosine phenol group near the O2 reduction site with the direct covalent link between the two groups. The structure suggests that the phenol group is the site for donating protons to the bound O2. Redox-coupled conformational change in an extramembrane loop indicates that an aspartate (Asp51) in the loop apart from the O2 reduction site is the site for proton pumping.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain

P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.     

Structural properties of the zinc site in Cu,Zn-superoxide dismutase; Perturbed angular correlation of gamma ray spectroscopy on the Cu, 111Cd-superoxide dismutase derivative

The Zn(II) site of the dimeric Cu(II),Zn(II)-superoxide dismutase from Saccharomyces cerevisiae has been examined by means of perturbed angular correlation of gamma rays (PAC) on the Cu(II),Cd(II)- and Cu(I),Cd(II)-superoxide dismutase. The PAC spectrum for the Cu(II),Cd(II) enzyme reveals two different, pH independent, coordination geometries for the Cd site. Removal of Cu(II) does not affect the PAC spectrum, which suggests that Cu(II) and Cd(II) do not share a common histidine side chain as ligand. The results are consistent with either an equilibrium between two coordination geometries for Cd(II) in each subunit or a difference in the structure of the Cd(II) site in the two subunits. In contrast, in the reduced enzyme only one structure is present, identical for the two subunits.     

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.     

Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.     

Kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II

The kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II has been studied over the pH range 5.2-9.9 at 25 degrees C and an ionic strength of 0.11 M. For all pH values, exponential decay curves are obtained for the reaction of compound II in the presence of ferrocyanide which yielded pseudo-first-order rate constants kobs. The spontaneous decay of compound II in the absence of ferrocyanide occurs at an appreciable rate which was measured independently and used in the data analysis. At all pH values two striking effects were observed when the rate of the decay reaction in the presence of ferrocyanide, kobs, was plotted against ferrocyanide concentration: a saturation effect and positive intercepts which are attributable to the spontaneous decay. The plots of kobs versus ferrocyanide concentration were analyzed in terms of the following parameters: a first-order rate constant k3,obs, a Michaelis constant Km,obs and a spontaneous-decay rate constant k4. The parameters k3,obs and Km,obs describe the reaction of compound II with ferrocyanide, independently of the spontaneous decay. The parameter k4 has only a small pH dependence, whereas plots of the logs of k3,obs and Km,obs versus pH have slopes of -1 at high pH. The major part of the pH dependence can be explained by the influence of a single heme-linked acid group in the LPO-compound-II-ferrocyanide complex.     

Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin: two vasopressin-like peptides.

The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.     

Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.     

Metal complexes of substituted pyrimidines

The interaction of Cu(II), Ni(II), Zn(II), Co(II), Mg(II) and Ca(II) ions with substituted pyrimidines, such as 2-mercaptopyrimidine, 4,5-diamino-6- hydroxypyrimidine and 2,4-diamino-6-hydroxypyrimidine, has been investigated by potentiometric studies. The proton dissociation constants of the ligands and the stability constants of the complexes containing 1:1 and 2:1 molar ratios of the ligand to the metal ions have been reported at 45°C and 0.1 M(KNO₃) ionic strength.