DNA affinity cleaving : Sequence specific cleavage of DNA by Distamycin-EDTA - Fe(II) and EDTA-distamycin Fe(II)

The attachment of EDTA· Fe(II) to distamycin changes the sequence specific DNA binding antibiotic into a sequence specific DNA cleaving molecule. We report the synthesis of EDTA-distamycin (ED) which has the metal chelator, EDTA, tethered to the carboxy terminus of the N-methylpyrrole tripeptide moiety of the antiobiotic, distamycin. EDTA-distamycin- Fe(II) (ED)·FeII at 10^-6M concentration efficiently cleaves pBR322 DNA (10^-5M in base pairs) in the presence of oxygen and dithiothreitol (DTT). Using Maxam-Gilbert sequencing gel analyses, we find that ED· Fe(II) affords DNA cleavage patterns of unequal intensity covering two to four contiguous base pairs adjacent to a five base pair site consisting of adenines (A) and thymines (T). The multiple cleavages at each site might be evidence for a diffusible oxidizing species, perhaps hydroxyl radical. The unequal intensity of cleavage on each side of the A + T site permit assignment of major and minor orientations of the tripeptide binding unit. A comparison of the cleavage specificity of ED· Fe(II) with distamycin-EDTA· Fe(II), (DE· Fe(II)) which has EDTA · Fe(II) attached to the amino terminus of the N-methylpyrrole tripeptide, shows DNA cleavage patterns at the same sites but with intensities of opposite polarity. Maxam-Gilbert sequencing el analysis of the DNA cleavage patterns by ED Fe(II) and DE Fe(II) on both DNA strands of a 381 se pair restriction fragment reveals asymmetric DNA cleavage patterns. Cleavage is shifted to the 3' de of each DNA strand. A model consistent with this cleavage pattern indicates one preferred binding te for ED Fe(II) and DE Fe(II) is 3'-TTTAA-5' with the “amino end” of the tripeptide oriented to e 3' end of the thymine rich strand. p]This “DNA affinity cleavage” method which consists of attaching cleaving functions to DNA binding molecules followed by DNA cleavage pattern analyses using Maxam-Gilbert sequencing gels may be a useful direct method for determining the binding site and orientation of small molecules on native DNA.     

Synthesis,characterization, catalase functions and DNA cleavage studies of new homo and heteronuclear Schiff base copper(II) complexes

A new tetradentate diimine–dioxime ligand containing a donor set of N₄, and its homo-, heterodinuclear and homotrinuclear copper(II) complexes were prepared and characterized on the basis of their elemental analysis, FT-IR, ¹H and ¹³C NMR spectra, molar conductivity and magnetic moment measurements. The extraction ability of N,N′′-bis[1-biphenyl-2-hydroxyimino-1-ethylidene]-diethylenetriamine was also evaluated in chloroform by using several transition metal picrates such as Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II) and Hg(II). It has been seen that the ligand shows strong binding ability toward the copper(II) ion. Moreover, the catalytic activities of the complexes for the disproportionation of hydrogen peroxide were investigated in the presence of imidazole. The synthesized complexes display efficiency in the disproportion reactions of hydrogen peroxide, producing water and dioxygen in catalase-like activity. The interaction between these complexes and DNA has also been investigated by agarose gel electrophoresis. We found that the homo- and heterodinuclear copper complexes can cleave supercoiled pBR322 DNA to nicked and linear forms. The dinuclear complexes including phenanthroline (2–4), with H₂O₂ as a co-oxidant, exhibited the strongest cleaving activity.     

Phosphate diesters and DNA hydrolysis by dinuclear Zn(II) complexes featuring a disulfide bridge and H-bond donors

The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn(II) complexes of BAPA are known to elicit the cooperation between the metal ion and the hydrogen-bond donating amino groups to greatly increase the rate of cleavage of phosphate diesters. The reactivity of the dinuclear complex 1·Zn(II)₂ toward bis-p-nitrophenyl phosphate and plasmid DNA was investigated and compared with that of reference complexes devoid of the disulfide bridge or of the hydrogen-bond donating amino groups. The dimetallic Zn(II) complex produces remarkable accelerations of the rate of cleavage of both the substrates accompanied by significant differences. In the case of BNP, the presence of the disulfide bridge does not lead to the improvement of the cooperative action of the two metal ions expected as the result of better preorganization. On the other hand, in the case of DNA the complex 1·Zn(II)₂ is much more reactive that the corresponding reference devoid of the disulfide bridge. Hence, different requisites must be fulfilled by a good catalyst for the cleavage of the two substrates. Moreover, binding studies with DNA indicated that the presence of two metal ions in the complex or of the pyridine amino groups, but not of the disulfide bridge, results into an enhanced affinity of the complexes toward this substrate.     

A Ferromagnetically Coupled (S=1) Peroxodicopper(II) Complex

Copper enzymes play important roles in the binding and activation of dioxygen in biological systems. Key copper/dioxygen intermediates have been identified and studied in synthetic analogues of the metalloprotein active sites, including the μ‐η²:η²‐peroxodicopper(II) motif relevant to type III dicopper proteins. Herein, we report the synthesis and characterization of a bioinspired dicopper system that forms a stable μ‐η¹:η¹‐peroxo complex whose Cu‐O‐O‐Cu torsion is constrained to around 90° by ligand design. This results in sizeable ferromagnetic coupling between the copper(II) ions, which is detected by magnetic measurements and HF‐EPR spectroscopy. The new dicopper peroxo system is the first with a triplet ground state, and it represents a snapshot of the initial stages of O₂ binding at type III dicopper sites.     

Cleavage of cyclopropene ring by means of transition metal salts

The oxidative ring cleavage of 1,3,3-trimethylcyclopropene (1) with Hg(OAc)₂ in methylene chloride gives 1,1-diacetoxy-2,3-dimethyl-2-butene (5). The reaction of 1 with TI(OAc)₃ or Pb(OAc)₄ gives in addition to 5, 3,3-diacetoxy-2-methylpropene and 2-acetoxy-4-methyl-1,3-pentadiene. These products are explained by assuming the addition of metal acetates onto the double bond of 1 followed by the ring cleavage to give vinylcarbene-metal acetate complexes or inverse ylides. The reaction of benzocyclopropene (14) with Hg(OAc)₂ affords bis(o-acetoxymethylphenyl)mercury as a single product. Cyclopropene ring cleavage of 14 effected by Cu(II) or Ag(l) acetate furnishes 9,10-dihydrophenanthrene and benzyl acetate. These products are ascribed to the intermediary vinylcarbene- metal acetate complexes produced from 14.     

Lewis Acid Coupled Electron Transfer of Metal–Oxygen Intermediates

Redox‐inactive metal ions and Brønsted acids that function as Lewis acids play pivotal roles in modulating the redox reactivity of metal–oxygen intermediates, such as metal–oxo and metal–peroxo complexes. The mechanisms of the oxidative C?H bond cleavage of toluene derivatives, sulfoxidation of thioanisole derivatives, and epoxidation of styrene derivatives by mononuclear nonheme iron(IV)–oxo complexes in the presence of triflic acid (HOTf) and Sc(OTf)₃ have been unified as rate‐determining electron transfer coupled with binding of Lewis acids (HOTf and Sc(OTf)₃) by iron(III)–oxo complexes. All logarithms of the observed second‐order rate constants of Lewis acid‐promoted oxidative C?H bond cleavage, sulfoxidation, and epoxidation reactions of iron(IV)–oxo complexes exhibit remarkably unified correlations with the driving forces of proton‐coupled electron transfer (PCET) and metal ion‐coupled electron transfer (MCET) in light of the Marcus theory of electron transfer when the differences in the formation constants of precursor complexes were taken into account. The binding of HOTf and Sc(OTf)₃ to the metal–oxo moiety has been confirmed for Mn^IV–oxo complexes. The enhancement of the electron‐transfer reactivity of metal–oxo complexes by binding of Lewis acids increases with increasing the Lewis acidity of redox‐inactive metal ions. Metal ions can also bind to mononuclear nonheme iron(III)–peroxo complexes, resulting in acceleration of the electron‐transfer reduction but deceleration of the electron‐transfer oxidation. Such a control on the reactivity of metal–oxygen intermediates by binding of Lewis acids provides valuable insight into the role of Ca²⁺ in the oxidation of water to dioxygen by the oxygen‐evolving complex in photosystem II.     

Ferrocene-conjugated copper(II) dipyridophenazine complex as a multifunctional model nuclease showing DNA cleavage in red light

The copper(II) complex [Cu(L)(dppz)](ClO₄)₂ (1) having a tripodal ligand ferrocenylmethylbis(2-pyridylmethylamine) (L) with a pendant ferrocenyl unit and a planar NN-donor dipyrido-[3,2-a:2′,3′-c]-phenazine (dppz) base is prepared and its DNA binding and cleavage properties studied. The complex is redox active showing cyclic voltammetric responses at 0.52 and –0.01 V vs. SCE due to Fe(III)/Fe(II) and Cu(II)/Cu(I) couples, respectively. The complex that binds to the major groove of DNA shows dual chemical nuclease activity involving both the metal centres. The complex displays efficient photo-induced DNA cleavage activity in visible laser light of 458 and 568 nm wavelengths forming cleavage active hydroxyl radicals. Significant DNA cleavage is also observed in red light of 647 nm within the photodynamic therapy (PDT) window.     

Oxidation of 3-hydroxyflavones in the presence of copper(I) and copper(II) chlorides

The reactions of 3-hydroxyflavones with dioxygen in the presence of CuCl and CuCl₂ have been studied. 2-Benzoyl-2-hydroxy-3(2H)-benzofuranone (2) was formed along with 2-hydroxybenzil (4), salicylic acid (5), benzoic acid (6), coumaronedione (8) and the depside (11). A copper(II) flavonolate complex has been also isolated.     

DNA binding and photocleavage specificities of a group of tricationic metalloporphyrins

The interactions of 5,10,15-tris(1-methylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatozinc(II) Zn[TMPyHP]³⁺ (2) along with Cu[TMPyHP]³⁺ (3), Co[TMPyHP]⁴⁺ (4), Mn[TMPyHP]⁴⁺ (5) and the free base porphyrin H₂[TMPyHP]³⁺ (1) with duplex DNA have been studied by using a combination of absorption, fluorescence titration, surface-enhanced Raman spectroscopy (SERS), induced circular dichroism (ICD) spectroscopy, thermal DNA denaturation, viscosity measurements as well as gel electrophoresis experiment. Their binding modes and intrinsic binding constants (K_b) to calf DNA (CT DNA) were comparatively studied and were found significantly influenced by different metals coordinated with the porphyrin plane. Except 3, which has four-coordination structure at the metal, all the metal derivatives showed non-intercalative DNA-binding mode and lower K_b than the free base porphyrin 1, most probably due to the steric hindrance results from the axial ligands of the inserted metals which are five or six-coordination structures. Meanwhile, the insertion of metals into cationic porphyrin greatly removed the self-aggregation of the metal-free porphyrins, and thus fully enhanced the singlet oxygen (¹O₂) productivities in the DNA photocleavage experiments. Therefore, these metalloporphyrins have comparable DNA cleavage ability with the free base porphyrin.     

Nickel‐substituted iron‐dependent cysteine dioxygenase: Implications for the dioxygenation activity of nickel model compounds

Reported here is a density functional theory study on the ability of Ni‐substituted iron‐dependent cysteine dioxygenase (CDO) to catalyze the oxidation of cysteine to cysteine sulfinic acid. The first steps of the commonly accepted mechanism for CDO, the O₂ activation mechanism, suggests the binding of O₂ to the metal ion (where redox isomerism takes place converting O₂ to ) followed by the attack of the distal oxygen atom on the cysteine sulfur—in line with most previous evidence. An alternative mechanism entailing the attack of the cysteine sulfur on the proximal oxygen atom of the dioxygen moiety to form a persulfenate intermediate without any redox exchange between the metal ion and the O₂ ligand, is supported by an X‐ray crystal structure showing a CDO with a bound cysteine persulfenate, and also supported by data on the oxidation of thiols catalyzed by Ni(II) compounds. Our results show that the O₂ activation mechanism with a Ni‐substituted active site follows the same pattern as native CDOs albeit with much higher energy barriers for the formation of the intermediates suggesting that the reaction might not be biologically feasible. Conversely, the immediate cleavage of the persulfenate S O bond in the alternative mechanism suggests that cysteine persulfenate might not be a true intermediate in catalytic cycle of CDOs.     

Proton reduction and dihydrogen oxidation on models of the [2Fe]H cluster of [Fe] hydrogenases. A density functional theory investigation

Density functional theory was used to compare reaction pathways for H2 formation and H+ reduction catalyzed by models of the binuclear cluster found in the active site of [Fe] hydrogenases. Terminal H+ binding to an Fe(I)-Fe(I) form, followed by monoelectron reduction and protonation of the di(thiomethyl)amine ligand, can conveniently lead to H2 formation and release, suggesting that this mechanism could be operative within the enzyme active site. However, a pathway that implies the initial formation of Fe(II)-Fe(II) mu-H species and release of H2 from an Fe(II)-Fe(I) form is characterized by only slightly less favored energy profiles. In both cases, H2 formation becomes less favored when taking into account the competition between CN and amine groups for H+ binding, an observation that can be relevant for the design of novel synthetic catalysts. H2 cleavage can take place on Fe(II)-Fe(II) redox species, in agreement with previous proposals [Fan, H.-J.; Hall, M. B. J. Am. Chem. Soc. 2001, 123, 3828] and, in complexes characterized by terminal CO groups, does not need the involvement of an external base. The step in H2 oxidation characterized by larger energy barriers corresponds to the second H+ extraction from the cluster, both considering Fe(II)-Fe(II) and Fe(II)-Fe(III) species. A comparison of the different reaction pathways reveals that H2 formation could involve only Fe(I)-Fe(I), Fe(II)-Fe(I), and Fe(II)-Fe(II) species, whereas Fe(III)-Fe(II) species might be relevant in H2 cleavage.     

A luminescence probe for measurements of electron-exchange rates in polymer films on electrodes

The effect of metal ions on the, reduction of 1,10-phenanthroline-5,6-quinone (1), 5,8-quinolinequinone (II) and 6,7-dichloro-5, 8-quinolinequinone (III) has been investigated in 50% dimethylsulfoxide+water solvent. 1 contains the 1,10-phenanthroline structure in both its quinone and hydroquinone forms, while II and III contain the 8-hydroxyquinoline structure in the hydroquinone forms. Complexation of the hydroquinones of II and III by metal ions causes positive shifts in the quinone half-wave potentials. These shifts have been used to calculate conditional formation constants for Pb²⁺(II) and Pb²⁺ and Zn²⁺(III). The quinone form of I binds strongly to Ni²⁺, Co²⁺ and Zn²⁺ but not to Ca²⁺. Mg²⁺. Mg₂₊. Mn²⁺ and Pb²⁺. With the latter four metals, binding to the hydroquinone from of I was detected and formation constants were determined. In addition to binding both the quinone and hydroquinone forms at the nitrogen atoms, Ni₂₊ Co²⁺ and Zn²⁺ formed complexes at the 1,2-dihydroxy site of the hydroquinone of I.     

Synthesis and in vitro biology of Co(II), Ni(II), Cu(II) and Zinc(II) complexes of functionalized beta-diketone bearing energy buried potential antibacterial and antiviral O,O pharmacophore sites

The clinically active functionalized β-diketones 1-(2′,4′-dihydroxyphenyl)-3-(2″-substitutedphenyl)-propane-1,3-dione (L₁)–(L₂) have been synthesized from Baker–Venkataraman transformation of 2,4-diaroyloxyacetophenones. Their transition metal complexes (1)–(8) have been prepared and characterized by physical, spectral and analytical data. The functionalized beta-diketone potentially acts as bidentate ligand and co-ordinate with the transition metal atom through beta-diketo system. The complexes have general formula [ML₂] where M = Co(II), Ni(II), Cu(II), Zinc(II) and L = ligand. The 1-(2′,4′-dihydroxyphenyl)-3-(2″-substitutedphenyl)-propane-1,3-dione and their transition metal complexes have been screened for in vitro antibacterial, antifungal and antioxidant bioassay. The biological activity data show that the transition metal complexes are more potent antibacterial, antifungal and antioxidant agents than the parent functionalized beta-diketone against different bacterial and fungal species. This constitutes a new group of compounds that can be used as potential metal derived drugs. Ultimately, here we can prompt about the use of metals for the drugs. The metal complexes were also studied for their thermogravimetric analyses.     

Metal‐Free Radical 5‐exo‐dig Cyclizations of Phenol‐Linked 1,6‐Enynes for the Synthesis of Carbonylated Benzofurans

A new metal‐free radical 5‐exo‐dig cyclization of phenol‐linked 1,6‐enynes with O₂, 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO), and tBuONO is described. With this general method, carbonylated benzofurans can be accessed through incorporation of two oxygen atoms into the product from O₂ and TEMPO through dioxygen activation and oxidative cleavage of the N? O bond, respectively.     

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4-His Fe(II) center and dioxygen. Herein, we use in-protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side-on fashion, as suggested from the experiment. The quintet Fe−O₂ complex with the side-on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate-limiting barrier of 10.0 kcal mol⁻¹. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate-limiting barrier (26.8 kcal mol⁻¹). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non-heme enzymes.     

Synthesis, crystal structure, electrochemistry and studies on protein binding, antioxidant and biocidal activities of Ni(II) and Co(II) hydrazone complexes

Bivalent transition metal hydrazone complexes of the composition [Ni(L₁)₂] (1), [Co(L₁)₂] (2), [Ni(L₂)₂] (3) and [Co(L₂)₂] (4) have been synthesised from the reactions of [MCl₂(PPh₃)₂] (where M = Ni or Co) with hydrazones derived from 2-acetyl pyridine and carboxylic acid hydrazides of benzhydrazide (HL₁) or thiophene-2-carboxylic acid hydrazide (HL₂), respectively. Structure of the ligands HL₁ and HL₂ and their corresponding complexes with Ni(II) and Co(II) ions were proposed based on the elemental analysis, infrared and ¹H NMR spectral methods. Single crystal X-ray diffraction study of complex 1 revealed a distorted octahedral geometry around the metal ion provided by two units of the ligand. To explore the potential medicinal value of the new complexes, binding interaction of all the complexes with bovine serum albumin (BSA) was studied at normal physiological conditions using fluorescence and UV-Vis spectral techniques. The number of binding sites (n) and binding constant (K_a) were calculated according to the double logarithm regression equation. The results of synchronous fluorescence spectrum showed that binding of metal hydrazones with BSA induced conformational changes in BSA. The in vitro antioxidant and antimicrobial potentials of the new chelates were also carried out.     

DNA Hydrolysis by Homo- and Heteronuclear Cu(II)–Ni(II) Complexes of Two Diester-type Ligands

Nucleolytic activities of novel mononuclear Cu(II), homo- and heterodinuclear Cu(II)–Ni(II) complexes with two diester-type ligands were investigated on pCYTEXP by neutral agarose gel electrophoresis. The analyses of the cleavage products obtained electrophoretically indicate that the examined complexes induce very similar conformational changes on supercoiled DNA by converting supercoiled form to nicked form. At concentrations greater than 100M, the complexes possessed effective nucleolytic activities for 10min of incubation time. However, their nucleolytic activities did not increase significantly with longer periods of incubation. The pH-nucleolytic activity profiles of the complexes differed significantly. Metal complex induced DNA cleavage was also tested for inhibition by various radical scavengers. It could be proposed from the data that diffusible intermediate oxidants are not involved in these reactions or they are not necessary for DNA cleavage since none of antioxidants inhibited DNA cleaving activities of the complexes.     

Reversible Coordination of Dioxygen by Tripodal Tetraamine Copper Complexes Incorporated in a Porous Silica Framework

The present study reports the synthesis and rational design of porous structured materials by using a templating method. A tetraethoxysilylated tripodal tetraamine (TREN) was covalently incorporated in a silica framework with a double imprint: A surfactant template and a metal ion imprint. The presence of a cationic surfactant (CTAB) endowed the material with a high porosity, and the tripodal or square‐pyramidal topology of the ligand was preserved thanks to the use of the silylated Cu^II complex. After removal of the surfactant and de‐metalation, the incorporated tetraamine was quantitatively complexed by CuCl₂ and the material has shown after thermal activation that a reversible binding of O₂ on the metal ions occurred. This chemisorption process was monitored by UV/Vis and EPR spectroscopies, and the Cu:O₂ adduct was postulated to be an end‐on μ‐η¹:η¹‐peroxodicopper(II) complex bridged by a chloride ion. The Cu^I‐active species, formed during the activation step, were fully recovered during several O₂ binding cycles. The high reactivity of the copper complexes and the room‐temperature stability of the dioxygen adduct were explained by the fine adaptability of the tripodal ligand to different geometries, the confinement of the active sites in the hybrid silica that protect them from degradation by a control of the metal‐ion microenvironment, as well as the short‐range lamellar order of the copper complexes in the framework.     

Synthesis,characterization, DNA binding,topoisomerase I inhibition,and antiproliferation activities of (di-<Emphasis Type="Italic">tert</Emphasis>-butylbipyridine) platinum(II) complexes

Two mononuclear Pt(II) complexes, Pt(dbbpy)Cl₂ (1) and [Pt(dbbpy)₂](PF₆)₂ (2) (dbbpy?=?4,4′-ditertbutyl-2,2′-biyridine) were synthesized and characterized by single-crystal X-ray diffraction analysis, elemental analysis, ¹H NMR, and ESI–MS. Their binding affinities for both double-stranded (DS) calf thymus DNA (ct-DNA) and G-quadruplex DNA (HT21 and BCL-2) were investigated. In addition to structural differences, complex 1 displayed higher binding affinity for DS ct-DNA, whereas positively charged complex 2 was selective for binding to G-quadruplex DNA over DS DNA. The time-dependent cleavage of supercoiled circular plasmid pBR322 DNA by 1 was observed using agarose gel electrophoresis, whereas complex 2 hardly cleaved DS DNA. Stabilization of G-quadruplex HT21 DNA by both complexes was assessed by PCR stop assays. Both complexes exhibited moderate activities for inhibition of topoisomerase I as well as modest antiproliferation activities toward cancer cells in CKK-8 assays.     

Theoretical and experimental investigation of the semiquinone forms of protocatechuic acid. The effect of manganese

Ten oxidized, oxygenated and dimeric forms of protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid, 3,4-DHBA) have been studied using DFT calculations (at the B3LYP/TZVP level of theory) and their structural and spectroscopic parameters (electronic transitions, NMR resonances) have been calculated. Combination with experimental results (under anaerobic or aerobic environment) determines the conditions for the existence of protonated, fully deprotonate and/or oxygenated semiquinones of PCA. Several energy optimized conformers containing manganese–(PCA–semiquinones) and water or/and peroxo-groups have been drawn (species 11–16) and their structural and spectroscopic properties have been calculated at the same level of theory. Experimental parallel to the theoretical results provide evidence for the existence of Mn(II)– and Mn(III)–[PCA–semiquinone] as well the conditions of dioxygen activation. Two of the blue solids (17 and 18) isolated from these solutions, have been characterized. Elemental analyzes, TGA, IR and ESR spectra support the formulation Mn₂(PCA)₂(O₂)(OH)₂(AcO)(ClO₄)₂(H₂O)₃ (17), and Mn₂(PCA)₂(O₂)₂(OH)₂(AcO)H₂O (18). Their ESR spectra, in solution (blue solutions), are almost identical and indicative of Mn(IV) existence. From the whole investigation, the activation of dioxygen by the PCA, its relocation on manganese and the oxidation of the metal ion have been provided.