New biocatalysts mimicking oxidative hemoproteins: Hemoabzymes

Catalytic antibodies with a metalloporphyrin cofactor or “hemoabzymes”, used as models for hemoproteins like peroxidases and cytochrome P450s, represent a promising route to catalysts tailored for selective oxidation reactions.The first strategy has been to produce anti-porphyrin antibodies, raised against various N-substituted- and meso-carboxyaryl-porphyrins, which led to monoclonal antibodies exhibiting, in the presence of the corresponding iron-porphyrin cofactor, a significant peroxidase activity. We ourselves obtained an artificial hemoprotein by associating a monoclonal antibody, 13G10, and its iron(III)-α,α,α,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin (Fe(ToCPP)) hapten, which exhibited a significant peroxidase activity. Biological studies suggested that in this antibody, a carboxylic acid side chain of the protein participated in the catalysis, but no amino acid residue acting as an axial ligand of the iron was detected. Therefore, to provide the iron atom with an axial ligand, we raised antibodies against microperoxidase 8, a heme octapeptide containing a histidine bound to the iron atom.This strategy was successful, as an antibody–microperoxidase 8 complex (3A3–MP8) led to the best k_cat/K_m ever reported for antibody–porphyrin complexes. The ability of the 3A3–MP8 complex to catalyze the selective oxidation of substrates was studied and it was found able to catalyze the regioselective nitration of aromatics by NO₂⁻/H₂O₂ as well as the stereoselective oxidation of sulfides like thioanisole by H₂O₂.Other strategies based on antibodies have to be developed to obtain more efficient biomimetic systems for cytochrome P450s. A first one could involve the modification of anti-substrate antibodies by covalent linkage of an iron(III)-porphyrin close to the binding site of the substrate, to obtain an artificial hemoprotein able to catalyze its regioselective oxidation.     

Direct Observation of an Oxepin from a Bacterial Cytochrome P450‐Catalyzed Oxidation

The cytochromes P450 are hemoproteins that catalyze a range of oxidative C?H functionalization reactions, including aliphatic and aromatic hydroxylation. These transformations are important in a range of biological contexts, including biosynthesis and xenobiotic biodegradation. Much work has been carried out on the mechanism of aliphatic hydroxylation, implicating hydrogen atom abstraction, but aromatic hydroxylation is postulated to proceed differently. One mechanism invokes as the key intermediate an arene oxide (and/or its oxepin tautomer). Conclusive isolation of this intermediate has remained elusive and, currently, direct formation of phenols from a Meisenheimer intermediate is believed to be favored. We report here the identification of a P450 [P450_cam (CYP101A1) and P450_cin (CYP176A1)]‐generated arene oxide as a product of in vitro oxidation of tert‐butylbenzene. Computations (CBS‐QB3) predict that the arene oxide and oxepin have similar stabilities to other arene oxides/oxepins implicated (but not detected) in P450‐mediated transformations, suggesting that arene oxides can be unstable terminal products of P450‐catalyzed aromatic oxidation that can explain the origin of some observed metabolites.     

Determination of iron-porphyrin-like complexes at nanomolar levels in seawater

A new method for the non-specific determination of iron-porphyrin-like complexes in natural waters has been developed. It is based on the chemiluminescent oxidation of the luminol in the presence of dioxygen (O₂) at pH 13. The method has been implemented in a FIA manifold that allowed the direct injection of seawater. The limit of detection is 0.11 nM of equivalent hemin (Fe-protoporphyrin IX). Fe²⁺, Fe³⁺, H₂O₂, siderophore (deferoxamin mesylate), humic acid and phytic acid did not interfere when they were present at the concentrations expected in seawater. Metal free porphyrin and Mg, Cu, Co porphyrin complexes did not induce a significant chemiluminescent signal. Poisoned unfiltered samples could be stored for several weeks before analyses. The new method was successfully applied to the determination of the Fe-porphyrin complexes contained in cultured phytoplankton and in natural samples.     

Oxidation of organic molecules in homogeneous aqueous solution catalyzed by hybrid biocatalysts (based on the Trojan Horse strategy)

New anionic metalloporphyrin–estradiol conjugates have been synthesized and fully characterized, and have been further associated to a monoclonal anti-estradiol antibody 7A3, to generate new artificial metalloenzymes following the so-called ‘Trojan Horse’ strategy. The spectroscopic characteristics and dissociation constants of these complexes were similar to those obtained for the artificial metalloproteins obtained by association of cationic metalloporphyrin–estradiol conjugates to 7A3. This demonstrates that the nature of the porphyrin substituents, anionic or cationic, had little influence on the association with the antibody that is mainly driven by the tight association of the estradiol anchor with the binding pocket of the antibody.These new biocatalysts appeared to have an interesting catalytic activity in oxidation reactions. The iron(III)–anionic-porphyrin–estradiol-antibody complexes were found able to catalyze the chemoselective and slightly enantioselective (ee = 10%) sulfoxidation of sulfides by H₂O₂. The Mn(III)–porphyrin–estradiol-antibody complexes were found to catalyze the oxidation of styrene by KHSO₅, the Mn(III)–cationic-porphyrin–estradiol-antibody complexes even showing the highest yields so far reported for the oxidation of styrene catalyzed by artificial metalloproteins. However, a lack of chemoselectivity and enantioselectivity was observed, which was probably due to a weak interaction of the metalloporphyrin cofactor with the binding pocket of antibody 7A3, as suggested by the similar UV–visible characteristics and catalytic activities obtained with both anionic and cationic porphyrins.     

Controllable stereoinversion in DNA-catalyzed olefin cyclopropanation via cofactor modification

The assembly of DNA with metal-complex cofactors can form promising biocatalysts for asymmetric reactions, although catalytic performance is typically limited by low enantioselectivities and stereo-control remains a challenge. Here, we engineer G-quadruplex-based DNA biocatalysts for an asymmetric cyclopropanation reaction, achieving enantiomeric excess (ee_trans) values of up to +91% with controllable stereoinversion, where the enantioselectivity switches to −72% ee_trans through modification of the Fe-porphyrin cofactor. Complementary circular dichroism, nuclear magnetic resonance, and fluorescence titration experiments show that the porphyrin ligand of the cofactor participates in the regulation of the catalytic enantioselectivity via a synergetic effect with DNA residues at the active site. These findings underline the important role of cofactor modification in DNA catalysis and thus pave the way for the rational engineering of DNA-based biocatalysts.

Cofactor modification in a DNA-catalyzed olefin cyclopropanation reaction enables controllable stereoinversion and achieves enantioselectivities of up to +91% and −72% ee_trans.     

Reaction Mechanism of Enzyme Studied by Pulse Radiolysis

By virtue of its ability to generate hydrated electrons (e_aq ^?) and various radicals as reductants, the pulse radiolysis technique has been employed for investigating the mechanism of action of peroxidase, cytochrome P-450, and cytochrome oxidase. The oxy forms of hemoproteins, such as myoglobin, peroxidase, and cytochrome P-450, were reduced by hydrated electrons to form the higher oxidation states of these hemoproteins. From these results, the reactive oxygen intermediate of cytochrome chrome P-450 is discussed. The reduction of cytochrome oxidase by the 1-methylnicotinamide radical was investigated. A decrease of the 830-nm band was detected due to the reduction of “visible” copper. After the first phase of the reduction of copper, the return of the 830-nm band corresponding to oxidation of copper was observed. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme α increased. This suggests that 1-methylnicotinamide radical reacts with the “visible” copper and subsequently flows to heme α by intramolecular migration.     

Porphyrin Nanorods Modified Glassy Carbon Electrode for the Electrocatalysis of Dioxygen,Methanol and Hydrazine

Porphyrin nanorods (PNR) were prepared by ionic self‐assembly of two oppositely charged porphyrin molecules consisting of free base meso‐tetraphenylsulfonate porphyrin (H₄TPPS₄^2?) and meso‐tetra(N‐methyl‐4‐pyridyl) porphyrin (MTMePyP⁴⁺M=Sn, Mn, In, Co). These consist of H₄TPPS₄^2?? SnTMePyP⁴⁺, H₄TPPS₄^2?? CoTMePyP⁴⁺, H₄TPPS₄^2?? InTMePyP⁴⁺ and H₄TPPS₄^2?? MnTMePyP⁴⁺ porphyrin nanorods. The absorption spectra and transmission electron microscopic (TEM) images of these structures were obtained. These porphyrin nanostructures were used to modify a glassy carbon electrode for the electrocatalytic reduction of oxygen, and the oxidation of hydrazine and methanol at low pH. The cyclic voltammogram of PNR‐modified GCE in pH 2 buffer solution has five irreversible processes, two distinct reduction processes and three oxidation processes. The porphyrin nanorods modified GCE produce good responses especially towards oxygen reduction at ?0.50 V vs. Ag|AgCl (3 M KCl). The process of electrocatalytic oxidation of methanol using PNR‐modified GCE begins at 0.71 V vs. Ag|AgCl (3 M KCl). The electrochemical oxidation of hydrazine began at around 0.36 V on H₄TPPS₄^2?? SnTMePyP⁴⁺ modified GCE. The GCE modified with H₄TPPS₄^2?? CoTMePyP⁴⁺ H₄TPPS₄^2?? InTMePyP⁴⁺ and H₄TPPS₄^2?? MnTMePyP⁴⁺ porphyrin nanorods began oxidizing hydrazine at 0.54 V, 0.59 V and 0.56 V, respectively.     

Stepwise Oxidations in a Cofacial Copper(II) Porphyrin Dimer: Through-Space Spin-Coupling and Interplay between Metal and Radical Spins

cis and trans-copper(II) porphyrin dimers have been synthesized, in which two Cu^II porphyrin macrocycles are bridged through a rigid ethene linker for possible through-space and through-bond spin-couplings between the paramagnetic Cu^II centers. It has been found that the two macrocycles come closer after 1 e⁻ oxidation, however, they move far apart upon further 1 e⁻ oxidation leading to transformation of the cis to the trans isomer. Detailed investigations are performed here on the interactions between the two porphyrin macrocycles, between two unpaired spins of closely spaced Cu^II centers, and also between the unpaired spins of metal and porphyrin π–cation radicals. Spectroscopic investigations along with the X-ray structure of the 2 e⁻-oxidized complex displayed strong electronic communications through the bridge between two porphyrin π–cation radicals. The counterion I₉⁻ is stabilized in an unusual trigonal-pyramidal structure in the 2 e⁻-oxidized complex in which the central iodide ion is bound with four iodine (I₂) molecules. Variable-temperature magnetic study revealed strong antiferromagnetic coupling between the two porphyrin π–cation radical spins (J_r–r) in the 2 e⁻-oxidized complex. DFT calculations suggest stabilization of the triplet state, which is also in good agreement with the experiment. Ab initio molecular dynamics allowed the variation of the structural details to be followed upon stepwise oxidations and also the final isomerization process including its associated energy barrier.     

Excitonically Coupled Cyclic BF2 Arrays of Calix[8]- and Calix[16]phyrin as Near-IR-Chromophores

Two giant calix[n]phyrin derivatives namely calix[8]- ( 4 ) and calix[16]phyrin ( 5 ), involving two and four BF₂ units, respectively, were prepared through the condensation of the bis-naphthobipyrrolylmethene-BF₂ complex ( 3 ) with pentafluorobenzaldehyde. Calix[n]phyrins 4 and 5 display extremely high extinction coefficients (3.67 and 4.82×10⁵ m ⁻¹ cm⁻¹, respectively) in the near-IR region, which was taken as initial evidence for strong excitonic coupling within these cyclic multi-chromophoric systems. Detailed insights into the effect of excitonic coupling dynamics on the electronic structure and photophysical properties of the macrocycles came from fluorescence, time-correlated single-photon counting (TCSPC) and transient absorption (TA) measurements. Support for these experimental findings came from theoretical studies. Theory and experiment confirmed that the coupling between the excitons depends on the specifics of the calix[n]phyrin structure, not just its size.     

Redox‐Driven Symmetry Change for Terbium(III) Bis(porphyrinato) Double‐Decker Complexes by the Azimuthal Rotation of the Porphyrin Macrocycles

Molecular structures for three oxidation forms (anion, radical, and cation) of terbium(III) bis(porphyrinato) double‐decker complexes have been systematically studied. We found that the redox state controls the azimuthal rotation angle (φ) between the two porphyrin macrocycles. For [Tb^III(tpp)₂]ⁿ (tpp: tetraphenylporphyrinato, n=?1, 0, and +1), φ decreases at each stage of the oxidation process. The decrease in φ is due to the higher steric repulsion between the phenyl rings on the porphyrin macrocycle and the β hydrogen atoms on the other porphyrin macrocycle, which results from the shorter interfacial distance between the two porphyrin macrocycles. Conversely, φ=45° for both [Tb^III(oep)₂]^?1 and [Tb^III(oep)₂]⁰ (oep: octaethylporphyrinato), but φ=36° for [Tb^III(oep)₂]⁺¹. Theoretical calculations suggest that the smaller azimuthal rotation angle of the cation form is due to the electronic interaction in the doubly oxidized ligand system.     

Greatly enhanced intermolecular π-dimer formation of a porphyrin trimer radical trications through multiple π bonds

A trefoil‐like porphyrin trimer linked by triphenylamine (TPA‐TPZn₃) was synthesized. A three‐electron oxidation of TPA‐TPZn₃ forms a radical trication (TPA‐TPZn₃³⁺), in which each porphyrin ring undergoes a one‐electron oxidation. The radical trication TPA‐TPZn₃³⁺ spontaneously dimerizes to afford (TPA‐TPZn₃)₂⁶⁺ in CH₂Cl₂. The characteristic charge‐resonance band due to the charge delocalization over the π system of (TPA‐TPZn₃)₂⁶⁺ was observed in the NIR region. The initial oxidation potential of TPA‐TPZn₃ is negatively shifted relative to that of the corresponding monomer porphyrin, which results from the stabilization of the oxidized state of TPA‐TPZn₃ associated with the dimerization. The thermodynamic parameters (i.e., ΔH, ΔS, and ΔG) for the formation of (TPA‐TPZn₃)₂⁶⁺ were determined by measuring Vis/NIR spectra at various temperatures. The formation constant of (TPA‐TPZn₃)₂⁶⁺ is significantly larger than that of the radical cation dimer of the corresponding monomer porphyrin (e.g., over 2000‐fold at 233 K). The electronic states were investigated using EPR spectroscopic analysis. The greatly enhanced dimerization of TPA‐TPZn₃³⁺ results from multiple π‐bond formation between the porphyrin radical cations.     

Excitonically Coupled Cyclic BF2 Arrays of Calix[8]‐ and Calix[16]phyrin as Near‐IR‐Chromophores

Two giant calix[n]phyrin derivatives namely calix[8]‐ ( 4 ) and calix[16]phyrin ( 5 ), involving two and four BF₂ units, respectively, were prepared through the condensation of the bis‐naphthobipyrrolylmethene‐BF₂ complex ( 3 ) with pentafluorobenzaldehyde. Calix[n]phyrins 4 and 5 display extremely high extinction coefficients (3.67 and 4.82×10⁵ m ^?1 cm^?1, respectively) in the near‐IR region, which was taken as initial evidence for strong excitonic coupling within these cyclic multi‐chromophoric systems. Detailed insights into the effect of excitonic coupling dynamics on the electronic structure and photophysical properties of the macrocycles came from fluorescence, time‐correlated single‐photon counting (TCSPC) and transient absorption (TA) measurements. Support for these experimental findings came from theoretical studies. Theory and experiment confirmed that the coupling between the excitons depends on the specifics of the calix[n]phyrin structure, not just its size.     

细胞色素P-450酶模型的研究:过氧化物的化学反应机理

含有铁卟啉化合物活性中心的细胞色素P-450和其它过氧化物酶具有重要的生物化学功能. 本文讨论了铁卟啉模型物模拟这些酶功能的反应机理. 通过与已知的过氧化物中氧氧键均裂机理的比较, 以及对大部分溶液反应的研究, 表明在过氧化物(包括过氧酸)与铁卟啉生成活性中间体( Fe=O^+.)的过程中, 氧氧键的异裂机理占主导地位. 过氧化物如同其它氧化剂受物(如烯烃)一样, 能快速地直接与 FeO^+反应. 以铁卟啉为催化剂的过氧化物反应体系为我们提供了研究模拟催化酶的人工模型体系.     

Adapting Synthetic Models of Heme/Cu Sites to Energy-Efficient Electrocatalytic Oxygen Reduction Reaction

In nature, cytochrome c oxidases catalyze the 4e⁻ oxygen reduction reaction (ORR) at the heme/Cu site, in which Cu^I is used to assist O₂ activation. Because of the thermodynamic barrier to generate Cu^I, synthetic Fe-porphyrin/Cu complexes usually show moderate electrocatalytic ORR activity. We herein report on a Co-corrole/Co complex 1-Co for energy-efficient electrocatalytic ORR. By hanging a Co^II ion over Co corrole, 1-Co realizes electrocatalytic 4e⁻ ORR with a half-wave potential of 0.89 V versus RHE, which is outstanding among corrole-based electrocatalysts. Notably, 1-Co outperforms Co corrole hanged with Cu^II or Zn^II. We revealed that the hanging Co^II ion can provide an electron to improve O₂ binding thermodynamically and dynamically, a function represented by the biological Cu^I ion of the heme/Cu site. This work is significant to present a remarkable ORR electrocatalyst and to show the vital role of a second-sphere redox-active metal ion in promoting O₂ binding and activation.     

Oxygenation of saturated and unsaturated hydrocarbons with sodium periodate catalyzed by manganese(III) tetra-arylporphyrins, to study the axial ligation of imidazole

Competitive oxygenation of cyclooctene and tetralin with sodium periodate catalyzed by Mn^(III)(TPP)OAc, TPP = meso-tetraphenylporphyrin; Mn^(III) (TNP)OAc, TNP =meso-tetrakis(1-naphthyl) porphyrin; Mn^(III) (TMP)OAc, TMP =meso-tetrakis(2,4,6-trimethyl-phenyl)porphyrin; Mn^(III) (TDCPP)OAc, TDCPP =meso-tetrakis(2,6-dichlorophenyl) porphyrin, and Mn^(III) (TPNMe₂-TFPP)OAc, TPNMe₂-TFPP =meso-tetrakis(para-NMe₂-tetrafluorophenyl)porphyrin, was carried out in the presence or absence of imidazole. This study showed that, in the absence of imidazole, selectivity for epoxide formation was high with electron-rich catalysts such as Mn(TPP)OAc, Mn(TNP)OAc and Mn(TMP)OAc, but low with electron-deficient catalysts such as Mn(TDCPP)OAc and Mn(TPNMe₂-TFPP)OAc. Presumably, not only the axial ligation of imidazole to the four-coordinate Mn(III)-center, but also the steric and electronic influences of aryl-substituents on the porphyrin periphery affect the selectivity of the catalytic oxidation reaction.     

Strategic Construction of Directly Linked Porphyrin–BODIPY Hybrids

A powerful and concise synthesis of directly linked porphyrin‐BODIPY hybrids has been demonstrated, which consists of condensation of directly linked meso ‐pyrroyl Ni^II‐porphyrin with arylaldehyde, oxidation with p ‐chloranil, and complexation with BF₃⋅Et₂O. Synthesized hybrids include porphyrin dimer 6Ni , trimers 8Ni , 9Ni , tetramer 12Ni , pentamer 16Ni , hexamer 13Ni , and nonamers 17Ni and 18Ni . The structures of 6Ni , 9Ni and 12Ni were unambiguously confirmed by X‐ray diffraction analysis. Some Ni^II porphyrins were effectively converted to the corresponding Zn^II porphryins. In these hybrids, the pigments are three‐dimensionally arranged with a face‐to‐face dimeric porphyrin unit in a well‐defined manner, featuring their potential as light‐harvesting antenna and functional hosts.     

Light- or oxidant-induced generation of free radicals in hemoproteins

Upon reactions with oxidants, hemoproteins retain their oxidizing equivalents not only in the heme iron, but also in the porphyrin or amino acid residues. HRP has a protein structure that stabilizes the porphyrin π-cation radical, which can easily be formed through various reactions. CCP reacts with H₂O₂ to form free radicals in amino acid residues as a catalytic intermediate species, which is stable in the absence of added electron donor. Ferric myoglobin apparently behaves as CCP, but its amino acid free radicals are unstable. These properties seem to be reflected on photooxidation of these hemoproteins.     

Tetrathiafulvalene Porphyrins

Four tetrathiafulvalene (TTF)‐annulated porphyrins 1 – 4 were synthesized and characterized. All contain a tetraphenylporphyrin (TPP) core onto which four, two, or one TTF subunits were annulated. Absorption and fluorescence spectroscopic studies together with electrochemical investigations reveal that interactions between the porphyrin system and the annulated TTF units take place in solution. The annulation of one or more TTF units to the porphyrin core has a profound effect on the reduction potentials associated with this latter framework, with positive shifts in the range of 0.105 to 0.355 V and 0.200 to 0.370 V for the first and second reduction potential, respectively, compared to the corresponding processes in the model compound TPP, 18 . The redox potentials for the first oxidation of the TTF units are considerably shifted in 4 (ΔE_ox¹=+0.285 V) and 2 (ΔE_ox¹=?0.140 V), whereas for 1 and 3 these potentials remain within the region expected for a normal TTF unit. Considerable changes in the second oxidation potential associated with the TTF subunits were seen for 2 (ΔE_ox¹=?0.085) and 3 (ΔE_ox¹=?0.175). The emission spectra of 1 – 4 revealed that the porphyrin fluorescence is almost quenched in the neutral state of the TTF‐annulated porphyrins, a finding that is consistent with substantial electron transfer taking place from the TTF subunits to the porphyrin core. Oxidation of the TTF unit(s) (TTF→TTF^.+) present in 1 – 4 leads to the emission intensity being restored.     

Functional chemical models of multielectron water oxidation in photosynthesis

The study of water oxidation by Mn _n ^IV clusters, which are functional chemical models of the manganese cofactor (enzyme that oxidizes water in photosystem II of natural photosynthesis) has demonstrated that a Mn₂^IV cluster oxidizes two water molecules to form one oxygen molecule, Mn₄^IVoxidizes four water molecules to form two O₂ molecules, and Mn₈^IVoxidizes eight water molecules to form four O₂ molecules. A Mn₆^IV cluster oxidizes six water molecules to two ozone molecules, whereas Mn₁₂^IV oxidizes twelve water molecules to four ozone molecules. The six-electron oxidation of water to ozone is also observed in photosynthesis in red and brown sea algae under conditions of water deficit. It is hypothesized that, in the case of water deficit in algae, the manganese cofactor with four manganese ions turns into the cofactor with six manganese ions.     

Enhanced Reactivities of Iron(IV)‐Oxo Porphyrin π‐Cation Radicals in Oxygenation Reactions by Electron‐Donating Axial Ligands

The proximal axial ligand in heme iron enzymes plays an important role in tuning the reactivities of iron(IV)‐oxo porphyrin π‐cation radicals in oxidation reactions. The present study reports the effects of axial ligands in olefin epoxidation, aromatic hydroxylation, alcohol oxidation, and alkane hydroxylation, by [(tmp)^+. Fe^IV(O)(p‐Y‐PyO)]⁺ ( 1 ‐Y) (tmp=meso‐tetramesitylporphyrin, p‐Y‐PyO=para‐substituted pyridine N‐oxides, and Y=OCH₃, CH₃, H, Cl). In all of the oxidation reactions, the reactivities of 1 ‐Y are found to follow the order 1 ‐OCH₃ > 1 ‐CH₃ > 1 ‐H > 1 ‐Cl; negative Hammett ρ values of ?1.4 to ?2.7 were obtained by plotting the reaction rates against the σ_p values of the substituents of p‐Y‐PyO. These results, as well as previous ones on the effect of anionic nucleophiles, show that iron(IV)‐oxo porphyrin π‐cation radicals bearing electron‐donating axial ligands are more reactive in oxo‐transfer and hydrogen‐atom abstraction reactions. These results are counterintuitive since iron(IV)‐oxo porphyrin π‐cation radicals are electrophilic species. Theoretical calculations of anionic and neutral ligands reproduced the counterintuitive experimental findings and elucidated the root cause of the axial ligand effects. Thus, in the case of anionic ligands, as the ligand becomes a better electron donor, it strengthens the FeO? H bond and thereby enhances its H‐abstraction activity. In addition, it weakens the Fe?O bond and encourages oxo‐transfer reactivity. Both are Bell–Evans–Polanyi effects, however, in a series of neutral ligands like p‐Y‐PyO, there is a relatively weak trend that appears to originate in two‐state reactivity (TSR). This combination of experiment and theory enabled us to elucidate the factors that control the reactivity patterns of iron(IV)‐oxo porphyrin π‐cation radicals in oxidation reactions and to resolve an enigmatic and fundamental problem.