ing a hydrogen atom from substrates with relatively weak X−H bonds (Figure 56). Such a situation was found in the catalytic cycle of ACCO, where cleavage of the N−H bond is facilitated by the ferrous ion capable of (partly) reducing the resulting N-based radical to an anion. In the catalytic cycle of HEPD and in the reaction of HppE with an enantiomer of the native substrate, a C−H bond at the carbon hosting a deprotonated alcohol group is severed by the superoxide. In these cases transfer of a hydrogen atom to the superoxide is coupled to one-electron reduction of Fe(III) to Fe(II), which yields aldehyde or ketone products. In a similar way a thioaldehyde is produced during the initial steps of the IPNS catalytic cycle. Figure 53. Reaction catalyzed by NDO. Figure 54. X-ray structure of the non-heme iron cofactor in the NDO active site (PDB 1O7G). Figure 55. Reaction mechanism for the dihydroxylation reaction catalyzed by NDO suggested on the basis of the results of a DFT study. Relative energy values in kilocalories per mole are given beneath the structures shown and above the arrows for the transition states connecting them. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3636 The most often encountered reaction of the Fe(III)−O2 species is an electrophilic attack on an electron-rich (co)substrate that yields an Fe(II) intermediate with a peroxide bridge between the ferrous ion and an organic molecule (Figure 57). In all examples shown in the figure the reactive spin state is a quintet with high-spin Fe(III) antiferromagnetically coupled to the superoxide radical. This particular electronic structure allows for a smooth two-electron reaction, leading to a ferrous intermediate featuring a high-spin Fe(II) ion, i.e., also lying on a quintet PES. When the substrates are easily one-electron-oxidized, as, for example, carotenoids or catecholates, already the ternary enzyme−dioxygen−organic substrate complex may contain an organic radical along with the superoxide anion stabilized by coordination to the metal (Figure 58). With proper (antiferromagnetic) alignment of the spins of the unpaired electrons on the two radicals, direct coupling between them proceeds with a straightforward formation of a new C−O bond. With the O2 ligand already protonated, several different reaction scenarios are usually plausible, and one of them, arguably the simplest, involves a transfer of the HOO group from the metal ion to the organic substrate (Figure 59). In the reaction of HEPD, it is the distal, i.e., originally protonated, oxygen atom that attacks the aldehyde group and the proton is transferred to the second (proximal) oxygen atom with the help of a phosphonic group of the substrate. In this reaction HOO acts as a nucleophile. In the case of HGD, the HOO ligand has a partial radical character, and hence, in its attack on the aromatic ring, it behaves as an electrophilic reagent. Moreover, in the HGD and ACO cases, it is the proximal oxygen atom that is directly transferred from the metal ion to the organic radical, i.e., the proton remains on the same oxygen atom throughout the reaction. When a catalytic reaction involves a homolytic O−O bond cleavage, one end of the peroxo group is in contact with the metal ion, and it is reduced to HO− or RO− when the O−O bond breaks (Figure 60). The electron required for the reduction is provided usually by the ferrous ion; in intradiol dioxygenases, which host Fe(III) in the active site, a tyrosinate ligand can serve as a reductant. Heterolytic cleavage of the O−O bond typically yields highvalent iron(IV)−oxo species, and such a reaction requires Fe(II), a deprotonated proximal oxygen atom, and usually also that the distal oxygen atom has a chance to develop a second covalent bond when the O−O gets broken (Figure 61). The bonding partner for the distal oxygen can be a hydrogen (PDHs, IPNS, ACCO) or a (co)substrate’s carbon (αKAOs, ACO, Dke1) atom. Heterolysis to Fe(IV)O usually proceeds on the quintet PES. Finally, heterolytic O−O bond cleavage may proceed without changing the oxidation state of the metal but instead with coupled two-electron oxidation of an organic substrate (Figure Figure 56. Examples of Fe(II)/Fe(III)−O2 reactions involving hydrogen atom abstraction. Only the most relevant fragment of the substrate is shown. Figure 57. Examples of Fe(III)−O2 reactions involving electrophilic attack and two-electron oxidation of an organic (co)substrate. Figure 58. Examples of Fe(II)/Fe(III)−O2 reactions involving radical coupling between the superoxide and an organic radical. Only the most relevant fragment of the substrate is shown. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3637 62). In both cases depicted in the figure, a hydroperoxo group is bound to high-spin Fe(III), and when theO−Obond cleaves, the OH group remains on iron and the other oxygen atom forms two covalent bonds with the organic substrate. This kind of heterolytic O−O cleavage requires that the donor orbital of the organic substrate, i.e., the one that provides two electrons for reduction of the peroxo group, has a good overlap with the O−O σ* orbital. 5.2. Dinuclear Non-Heme Iron Enzymes 5.2.1. Methane Monooxygenase. MMO is an enzyme which inserts one oxygen from O2 into methane to form methanol. The active site of the soluble form of MMO is shown in Figure 63. It contains an iron dimer complex linked by oxygen-derived ligands and has four glutamates and two histidines. Due to the presence of a well-resolved X-ray structure, and the technical importance of the reaction catalyzed, this was actually the first redox-active enzymemechanism that was treated with the cluster model using modern DFT functionals in 1997. Several groups were active at an early stage, and the most essential parts of the reaction mechanism were determined more than a decade ago. A comprehensive review of this development was written by Friesner et al. In short, the active species (compound Q) has a diamond core structure with two bridging oxo groups and is in an Fe2(IV,IV) state, one of themost oxidized species in nature. One of the oxo groups of compound Q activates nethane by an abstraction of one hydrogen atom. The TS is linear in C···H···O to form an Fe2(III,IV) state and amethyl radical. The loss of entropy at the TS is a large part of the barrier. In the second step, the methyl radical recombines with the bridging hydroxide formed in the first step. The TS for this step was first located by Basch et al. This rebound mechanism was criticized by interpretations of radical clock experiments, which appeared to show that there could not be a sufficiently long-lived alkyl radical to be consistent with a two-step mechanism. A concerted mechanism with simultaneous cleavage of the C−H bond and formation of the C−O bond was therefore suggested. Several explanations were suggested to resolve this apparent discrepancy between experiment and theory. In one of them, it was concluded that the radical clock probe molecules Figure 59. Examples of HOO transfer reactions. Only the most relevant fragment of the substrate is shown. Figure 60. Examples of O−O bond homolysis facilitated by oneelectron reduction of the proximal oxygen atom. Only the most relevant fragment of the substrate is shown. Figure 61. Examples of heterolytic O−O bond cleavage yielding iron(IV)−oxo species. Figure 62. Examples of heterolytic O−Obond cleavage proceeding with direct two-electron oxidation of the organic molecule. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3638 were chemically so different from methane that different mechanisms were likely. The probes are much easier to ionize and could form cations instead of radicals. Another suggestion was a so-called two-channel mechanism of the dynamics involving a bound radical intermediate. A third possibility could be something analogous to the two-state reactivity mechanism suggested for P450 to explain similar discrepancies in that case, but this has not been tested. In summary, a concerted mechanism, as suggested by the experiments, has never been found in DFT modeling calculations for methane hydroxylation by MMO, at least when reasonable models have been tried, and the suggestion has therefore not survived. In the initial phase of the MMO studies, there were problems converging to proper electronic states. These problems were solved by Friesner et al., who were able to obtain the correct antiferromagnetic coupling of compound Q with two high-spin irons. A state of key importance is also the first intermediate after Q, with an electronic structure characterized as Fe2(III,IV)− O•, discussed further below in connection with mixedMn−Fe dimers. It was found that already at the TS for hydrogen abstraction the iron dimer is in an Fe2(III,IV) state as it is in the product of this step. The bridging oxygen radical would then act as a hydrogen atom abstractor. At the TS, the spins are divided between a bridging oxo ligand and the methyl, while the iron spins stay essentially constant from the Fe2(III,IV)−O state to the product. The O−O bond cleavage to reach compound Q is also a significant step in the catalytic cycle. Again, several groups were involved in studying this step at an early stage. There was essential agreement among these studies on the mechanism. First, a peroxide (compound P) is formed between the two irons in an Fe2(III,III) state. Several different structures of P are nearly degenerate. In the TS for the O−O cleavage, the oxygens are symmetric, but only one of the irons is redox-active. In the final Figure 63. Active site of methane monooxygenase. Figure 64. R1 and R2 proteins in RNR. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3639 stage, the other iron also changes its oxidation state to IV, and compound Q is formed. In the study by Friesner et al., 

Quantum chemical studies of mechanisms for metalloenzymes.

Mechanism of extradiol aromatic ring-cleaving dioxygenases

A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus (Penicillium stipitatum) and investigated by targeted gene knockout A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group The tropA gene was characterized by heterologous expression in Aspergillus oryzae, whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group Comparison of the T stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone

https://www.pnas.org/content/pnas/109/20/7642.full.pdf

Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

This review is the first to collate and summarize main data on named and unnamed rearrangement reactions of peroxides. It should be noted, that in the chemistry of peroxides two types of processes are considered under the term rearrangements. These are conventional rearrangements occurring with the retention of the molecular weight and transformations of one of the peroxide moieties after O-O-bond cleavage. Detailed information about the Baeyer-Villiger, Criegee, Hock, Kornblum-DeLaMare, Dakin, Elbs, Schenck, Smith, Wieland, and Story reactions is given. Unnamed rearrangements of organic peroxides and related processes are also analyzed. The rearrangements and related processes of important natural and synthetic peroxides are discussed separately.

https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-12-162.pdf

Rearrangements of organic peroxides and related processes

Molecular oxygen is utilized in numerous metabolic pathways fundamental for life. Mononuclear nonheme iron-dependent oxygenase enzymes are well known for their involvement in some of these pathways, activating O2 so that oxygen atoms can be incorporated into their primary substrates. These reactions often initiate pathways that allow organisms to use stable organic molecules as sources of carbon and energy for growth. From the myriad of reactions in which these enzymes are involved, this perspective recounts the general mechanisms of aromatic dihydroxylation and oxidative ring cleavage, both of which are ubiquitous chemical reactions found in life-sustaining processes. The organic substrate provides all four electrons required for oxygen activation and insertion in the reactions mediated by extradiol and intradiol ring-cleaving catechol dioxygenases. In contrast, two of the electrons are provided by NADH in the cis-dihydroxylation mechanism of Rieske dioxygenases. The catalytic nonheme Fe center, with the aid of active site residues, facilitates these electron transfers to O2 as key elements of the activation processes. This review discusses some general questions for the catalytic strategies of oxygen activation and insertion into aromatic compounds employed by mononuclear nonheme iron-dependent dioxygenases. These include: (1) how oxygen is activated, (2) whether there are common intermediates before oxygen transfer to the aromatic substrate, and (3) are these key intermediates unique to mononuclear nonheme iron dioxygenases?

/pdf/oxygen-activation-by-mononuclear-nonheme-iron-dioxygenases-2g5l66jvw3.pdf

Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics.

Three mechanistic probes were used to investigate whether the Criegee rearrangement step of catechol 1,2-dioxygenase (CatA) from Acinetobacter sp. proceeds via a direct 1,2-acyl migration, via homolytic O-O cleavage, or via a benzene oxide-oxepin rearrangement. Incubation of CatA with 3-chloroperoxybenzoic acid led to the formation of a 9:1 mixture of 2-chlorophenol and 3-chlorophenol, via a mechanism involving O-O homolytic cleavage. Incubation of CatA with 2-hydroperoxy-2-methylcyclohexanone led to formation of 5,6-diketoheptan-1-ol, also consistent with an O-O homolytic cleavage mechanism, and not consistent with a direct 1,2-acyl migration. No reaction product was isolated from incubation of CatA with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone, an analogue that is able to undergo the benzene oxide-oxepin rearrangement, but not able to undergo O-O homolytic cleavage. In contrast, incubation of extradiol dioxygenase MhpB from Escherichia coli with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone led to the formation of a 2-tropolone ring expansion product, consistent with a direct 1,2-alkenyl migration for extradiol cleavage. Taken together, the results imply different mechanisms for the Criegee rearrangement steps of intradiol and extradiol catechol dioxygenases: a direct 1,2-alkenyl migration for extradiol cleavage and an O-O homolytic cleavage mechanism for intradiol cleavage.

Evidence from mechanistic probes for distinct hydroperoxide rearrangement mechanisms in the intradiol and extradiol catechol dioxygenases.

Substituted 2-tropolone natural products, are found in plants and fungi. Their biosynthesis is thought to occur by ring expansion from a cyclohexadienone precursor, but this reaction has not previously been demonstrated experimentally. Treatment of 6-hydroxy-6-hydroxymethylcyclohexa-2,4-dienone with the non-haem iron(II)-dependent extradiol catechol dioxygenase MhpB from Escherichia coli results in the formation of the 2-tropolone ring-expansion product through a pinacol-type rearrangement. Three further substituted cyclohexa-2,4-dienone analogues were prepared, and treatment of each analogue was found to give the substituted 2-tropolone ring-expansion product. This ring expansion could also be effected nonenzymatically by treatment with. 1,4,7-triazacyclonone and FeCl2. This is a novel transformation for non-haem iron-dependent enzymes, and this is the first experimental demonstration of the proposed ring-expansion reaction in tropolone biosynthesis.

Meite Xin

Papers

Evidence from mechanistic probes for distinct hydroperoxide rearrangement mechanisms in the intradiol and extradiol catechol dioxygenases.

Biomimetic Formation of 2-Tropolones by Dioxygenase-Catalysed Ring Expansion of Substituted 2,4-Cyclohexadienones