Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content--order restored

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2*- in the presence of Cu,Zn-SOD. We determined that k(cat) = (2.82 +/- 0.02) x 10(9) M(-1) s(-1) at low ionic strength (2 < I < 15 mM) and (1.30 +/- 0.02) x 10(9) M(-1) s(-1) in the presence of 50 mM phosphate at pH 7.8 (I = approximately 150 mM), which are about twice higher than those reported by Michel et al. We also determined k(cat) by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2*-.     

Mitochondrial chemiluminescence elicited by acetaldehyde

Acetaldehyde-dependent chemiluminescence has been found to be a sensitive technique for the study of superoxide and hydrogen peroxide formation in beef heart mitochondria. The system responds to ATP and antimycin A with increased emission intensities and to ADP and rotenone with decreased intensities, indicating that the chemiluminescence reflects the energy status of the mitochondrion. These effects are based on the ability of acetaldehyde to react with superoxide and hydrogen peroxide to form metastable intermediates which decay spontaneously with the emission of light. Additionally, these intermediates can react with cyanide to give alternative products which can also decay with the emission of light, the cyanide-evokable chemiluminescence. The interaction of acetaldehyde with mitochondria is complex because acetaldehyde can serve as a hydrogen source for NADH and as an inhibitor (at high concentration) of electron transport, and appears to be a reducing agent for a heat-stable site that autoxidatively generates HOOH from O₂ ^–·. Inasmuch as acetaldehyde is a metabolite of ethanol, this broad spectrum of reactivity may play a role in the hepatic and cardiac toxicity that is associated with alcoholism. The heat-stable site that generates HOOH from O₂ ^–· has been studied further and appears to contain vicinal dithiol which is primarily responsible for the cyanide-evokable chemiluminescence.The work reported in this paper was carried out by Erin E. Boh in partial fulfillment of the requirements for the Doctor of Philosophy degree.     

Spectroscopic analysis of the trinuclear cluster in the Fet3 protein from yeast, a multinuclear copper oxidase

The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.     

Synthesis,structure and catecholase activity of dinuclear copper and zinc complexes with an N(3)-ligand

The preparation and characterization of dinuclear [M(II)(dbcat)(idpa)](2) (M[double bond]Zn (1), Cu (3); dbcat[double bond]3,5-di-tert-butylcatecholate; idpa[double bond]3,3'-iminobis(N,N-dimethylpropylamine)) complexes are described. Crystallographic characterization of the complex [Cu(II)(dbcat)(idpa)](2) has shown that the co-ordination geometry around copper(II) ions is distorted square pyramidal (triclinic, P-1, a=10.576(1) A, b=11.927(1) A, c=12.621(1) A, alpha=77.89(1) degrees, beta=88.65(1) degrees, gamma=70.21(1) degrees, V=1462.7(2) A(3), Z=2, R=0.0387). Both 1 and 3 were suitable catalysts for the catalytic oxidation of dbcatH(2) to dtbq (dtbq=3,5-di-tert-butyl-1,2-benzoquinone) with dioxygen at ambient conditions in good yields. However, on the basis of kinetic studies the copper- and zinc-catalyzed reactions showed different mechanisms. In the first case valence tautomerism [Cu(II)(dbcat)(idpa)]<==>[Cu(I)(dbsq)(idpa)] precedes the reaction with O(2), while with the zinc complex metal-bound catecholate reacts directly with O(2) with the formation of free superoxide anion.     

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     

Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition

In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.     

Copper-catalyzed cleavage of DNA by arenes

DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested.     

A novel mixed valence form of Rhus vernicifera laccase and its reaction with dioxygen to give a peroxide intermediate bound to the trinuclear center

Rhus vernicifera laccase, in a novel mixed valence state [T1oxT23red: type 1 Cu as Cu(II), and type 2 and 3 Cus as Cu(I)], was formed by reacting Cu(I) on the type 2 Cu-depleted laccase [T1oxT3red: type 1 Cu as Cu(II) and type 3 Cus as Cu(I)] under argon. Contrary to T1oxT3red, T1oxT23red was highly reactive with dioxygen, and gave the three transient bands at 340, 475, and 680 nm due to the two-electron reduced form of dioxygen [charge transfer bands from peroxide to Cu(II)]. The first order decays were highly dependent on pH, which led to the successful detection of the intermediate for ca. 2 h at pH 7.5. Another mixed valence derivative, T12oxT3red [type 1 and type 2 Cus as Cu(II), and type 3 Cus as Cu(I)] prepared through the action of Cu(II) on T1oxT3red was not reactive with dioxygen, but showed high enzyme activity as to the oxidation of N,N-dimethyl-p-phenylenediamine. The whole reaction mechanism of the reduction of dioxygen by laccase was proposed based on the present results together with data for the former detection and characterization of the three-electron reduced form of dioxygen [Huang, H. et al. (1999) J. Biol. Chem. 274, 46, 32718-32724].     

Copper- and Arene-Catalyzed Cleavage of DNA

DNA was found to be cleaved by arenes and copper(II) salts in neutral solutions. The efficiency of this reaction is comparable with the DNA cleavage by such systems as Cu(II)–phenanthroline and Cu(II)–ascorbic acid in efficiency, but, unlike them, it does not require the presence of an exogenous reducing agent or hydrogen peroxide. The Cu²⁺–arene system does not cleave DNA under anaerobic conditions. Catalase, sodium azide as well as bathocuproine, a specific chelator of Cu(I), completely inhibit the reaction. Our results suggest that Cu(I) ions, superoxide radical and singlet oxygen participate in this reaction. It was shown by EPR and spin traps that the reaction proceeds with the formation of alkoxyl radicals capable of inducing breaks in DNA molecules. An efficient cleavage of DNA in the Cu(II)–o-bromobenzoic acid system requires the generation of radicals under the conditions of formation of a specific copper–DNA–o-bromobenzoic acid complex, in which copper ions are likely to be coordinated with oxygen atoms of the DNA phosphate groups.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V and IX with complex fluorides, chlorides and cyanides

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V and the tumour associated, transmembrane hCA IX, with complex anions incorporating fluoride, chloride and cyanide, as well as B(III), Si(IV), P(V), As(V), Al(III), Fe(II), Fe(III), Pd(II), Pt(II), Pt(IV), Cu(I), Ag(I), Au(I) and Nb(V) species has been investigated. Apparently, the most important factors influencing activity of these complexes are the nature of the central metal ion/element, and its charge. Geometry of these compounds appears to be less important, since both linear, tetrahedral, octahedral as well as pentagonal bipyramidal derivatives led to effective inhibitors. However, the five isozymes showed very different affinities for these anion inhibitors. The best hCA I inhibitors were cyanide, dicyanocuprate and dicyanoaurate (K(I)s in the range of 0.5-7.7 microM), whereas the least effective were fluoride and hexafluoroarsenate. The best hCA II inhibitors were cyanide, hexafluoroferrate and tetrachloroplatinate (K(I)s in the range of 0.02-0.51 mM), whereas the most ineffective ones were fluoride, hexafluoroaluminate and chloride. The best hCA IV inhibitors were dicyanocuprate (K(I) of 9.8 microM) and hexacyanoferrate(II) (K(I) of 10.0 microM), whereas the worst ones were tetrafluoroborate and hexafluoroaluminate (K(I)s in the range of 124-126 mM). The most effective hCA V inhibitors were cyanide, heptafluoroniobate and dicyanocuprate (K(I)s in the range of 0.015-0.79 mM), whereas the most ineffective ones were fluoride, chloride and tetrafluoroborate (K(I)s in the range of 143-241 mM). The best hCA IX inhibitors were on the other hand cyanide, heptafluoroniobate and dicyanoargentate (K(I)s in the range of 4 microM-0.33 mM), whereas the worst ones were hexacyanoferrate(III) and hexacyanoferrate(II).     

A kinetic study on the copper-albumin catalyzed oxidation of ascorbate

Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM^–1 min^–1 was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent K_m-value of 0.89 mM, and a catalytic constant of 0.066 M O₂/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H₂O₂ in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.     

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.     

Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design

Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site. A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis. These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized. We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2). The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not. We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers. Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin.     

Structural variety of copper(II)-peroxide adducts and its relevance to DNA cleavage

The reactivity of copper (II) compounds with several tetradentate ligands towards some spin-trapping reagents was studied in the presence of hydrogen peroxide. The compounds used in this study are roughly divided into two groups based on the reactivity towards 2,2,6,6-tetramethyl-4-piperidinol (and also 2,2,6,6-tetramethyl-4-piperidone), which are trapping agents for singlet oxygen. 1O2(1deltag); The A-group compounds exhibited a high activity to form the corresponding nitrone radical, which was detected by ESR spectroscopy, but corresponding activity of the B-group compounds was very low. The A-group compounds defined as above exhibited high activity for cleavage of DNA (supercoiled) Form I) in the presence of hydrogen peroxide, yielding DNA Form II (relaxed circular) or Form III (linear duplex) under our experimental conditions ([Cu (II)] = 0.1 approximately 0.5 mM). On the other hand, the B-group compounds effected complete degradation of the DNA (double-strand scission) under the same experimental conditions, formation of Form II or Form III DNA was negligible. Two different DNA cleavage patterns observed for A- and B-group compounds were elucidated by the different structural property of the copper (II)-peroxide adducts, which is controlled by the interaction through both DNA and the peripheral group of the ligand system.     

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.     

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the CH bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the "oxo-transfer": (i) the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1, recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421-4430.]; (ii) the most frequently used model complex, bis(mu-oxo)Cu(III)(2) complex 2; and (iii) the mixed-valence bis(mu-oxo)Cu(II)Cu(III) complex 3. The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a "singlet" potential surface and a "singlet oxene" is directly inserted across a CH bond in a concerted manner. Kinetic isotope effects (k(H)/k(D) or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5-6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3.     

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.     

Syntheses and characterization of copper complexes with the ligand 2-aminoethyl(2-pyridylmethyl)-1,2-ethanediamine (apme)

Copper(I)/(II) complexes with the ligand 2-aminoethyl(2-pyridylmethyl)1,2-ethanediamine (apme, abbreviated as PDT in the literature as well) were prepared and characterized. Crystal structures of the copper(I) complexes, [Cu₂(apme)₂]X₂ (1, 2; X = ClO₄, CF₃SO₃), showed that they are dinuclear, in contrast to the trigonal bipyramidal copper(II) complexes [Cu(apme)Cl]BPh₄ (3) and [Cu(apme)(DMF)](BPh₄)₂ (4). 1 and 2 could be investigated in solution by NMR spectroscopy and 3 and 4 by cyclovoltammetry. From the results of these studies it is clear that in solution equilibria between the dinuclear complexes 1/2 and another species exist, most likely the monomeric [Cu(apme)CH₃CN]⁺. Time-resolved UV/vis spectra at low temperatures allowed the spectroscopic detection of dioxygen adduct complexes as reactive intermediates during the oxidation of 1/2 with dioxygen that seem to play an important role in copper enzymes such as peptidylglycine--hydroxylating monooxygenase (PHM).