Ferryl intermediates

Numerous studies have shown that oxidation of a wide range of AH2 by HRP in the presence of H202 is characterized by a loss of enzyme activity. It is now well established that HRP is inactivated by H202.32 Because the final step (Equation 17.4), during which the oxidized ferryl intermediate is... 

Catalases and peroxidases both promote H2O2 reduction by mechanisms that involve ferryl intermediates. Catalases differ from peroxidases by their ability to use H2O2 both as an electron acceptor and as donor, thus catalysing the disproportionation reaction (catalatic activity) (equation 1) ... 

Peroxidases (EC 1.11.1.7). Peroxidases are hemoproteins, produced mainly by microorganisms and plants, which catalyze oxidation of the recalcitrant nonphenolic lignin units in the presence of hydrogen peroxide (Duran and Esposito, 2000). This is possible because of the formation of a high redox potential oxo-ferryl intermediate during the reaction of the heme cofactor with H202 (Martinez et al., 2005). Dubey et al. (1998) studied the polymerization of catechol by plant peroxidases and found that the resultant polymers consisted of phenylene and oxyphenylene units (Figure 2.14). 

Although ferryl intermediates of horseradish peroxidase and microperoxidase-8 have been produced in reactions with photogenerated [Ru(bpy)3]3+ [5], analogous experiments with P450s were unsuccessful, presumably due to the inefficiency of electron transfer from the buried heme active site through the protein backbone [6]. Photoactive molecular wires (sometimes referred to as metal-diimine wires, sensitizer-tethered substrates, or electron tunneling wires) were developed to circumvent this problem by providing a direct ET pathway between [Ru(bpy)3]3+ and the heme. These molecular wires, which combine the excellent photophysical properties of metal-diimine complexes... 

Although the peroxide shunt path to a ferryl intermediate is an attractive hypothesis, there is very little data to support the idea that the species obtained by this path are the same as, or similar to, that... 

Haem proteins that react with oxygen also utilise ferryl intermediates. Fig. 4 compares the (proposed) reaction mechanisms of cytochrome oxidase and cytochrome P-450 with those of peroxidases and catalases. As can be seen, several of the reaction intermediates have the same oxidation states (although the protonation steps and stage at which H2O is released may be different). However, in contrast to peroxidases, oxidases must react with molecular oxygen, and this requires a reaction cycle that includes Fe11. 

Fig. 5. Mechanism of action of dinuclear non-haem iron enzymes utilising ferryl intermediates. Mechanisms for ribonucleotide reductase and methane mono-oxygenase adapted from that of Que [72]. Compound I and compound II define intermediates at the same oxidation state as the equivalent peroxidase intermediate (see Fig. 2). X is an unknown group suggested to bridge between the two iron atoms and form a cation radical. The nature of the electron required for the reduction of ribonucleotide reductase compound II is not clear - it is possible that this intermediate can also oxidise tyrosine [72].

In analogy with peroxidase/P-450 mechanisms it is possible to devise a mechanism for these reactions that involve ferryl intermediates (Fig. 5). In favour of these analogies is the fact that H2O2 will act as an electron and oxygen donor to the di-ferric form of both enzymes, bypassing the need for a reductant and molecular oxygen [73-75]. However, it must be stressed that these mechanisms are still controversial [76]. Although the compound II-equivalent intermediate... 

Several of the proteins with ferryl intermediates have been crystalised at sufficient resolution to allow the elucidation of their 3-dimensional structure. These include cytochrome c peroxidase [95], horseradish peroxidase [96], catalase [97], myeloperoxidase [98], ribonucleotide reductase [99], cytochrome P-450 [100] and myoglobin [101]. Of these only cytochrome c peroxidase has proved stable enough to crystallise with the iron in the ferryl form [26]. High-resolution structures exist for small FeIV model compounds, both in the presence [102] and absence [7,8] of an Fe=0 bond. These compounds can have sulphur, nitrogen and chloride ligation to the iron and the iron can be five [7,8] or six [8] coordinate. 

The knowledge of these unique short iron-oxygen distances in ferryl compounds aids in identifying whether a compound has a ferryl structure. Thus the 580 nm compound formed upon addition of peroxide to mitochondrial cytochrome c oxidase has an iron-to-oxygen distance of 1.7 A, suggesting that it is a ferryl intermediate [117]. Of the two haem iron atoms in this molecule, one is unreactive to peroxide. Therefore it is possible to analyse an EXAFS spectrum of the peroxide-treated enzyme minus the spectrum of the untreated enzyme to determine this distance. However, clearly such difference EXAFS spectra will have increased errors associated with the estimate of iron-oxygen distances. 

There are no reported optical spectra attributed to FeIV in non-haem iron proteins suggested to have ferryl intermediates. However, a low-intensity peak at about 600 nm is observed in a model non-haem ferryl compound generated by the addition of H202to a (p-oxo) diferric complex [130],... 

One advantage of Raman spectroscopy is that it is relatively easy to perform time-resolved spectra on a sub-millisecond time scale. Therefore it is possible to obtain spectra of a reaction intermediate that decays rapidly. For example, coupling resonance Raman to flash photolysis resulted in the detection of the much-hypothesised ferryl intermediate in cytochrome c oxidase [207]. 

Progressive fibro-proliferative diseases (e.g. liver cirrhosis, pulmonary fibrosis, rheumatoid arthritis) result in a dramatic increase in collagen synthesis [227], This is preceded by inflammation that correlates with an increased activity of proline and lysine hydroxylase [228], Although they are unlikely to be the primary initiators of these diseases the increased activities of these enzymes may cause other problems. For example, in vitro the enzyme can turn over in the absence of a peptide substrate (but the presence of the 2-oxoglutarate cofactor). In this case stoichiometric amounts of ascorbate are required, probably to reduce the ferryl ion back to ferrous [229]. In vivo, lower concentrations of ascorbate are utilised [229,230], possibly to reactivate the enzyme after a non-productive activation (for example in the presence of a peptide that can bind to the active site, but cannot be hydroxylated). As the amount of proline-hydroxylase activity increases in the fibro-proliferative diseases, the concentration of ascorbate might not be sufficient to reduce these inactive complexes, resulting in the formation of potentially reactive ferryl intermediates. 

All haem proteins that utilise ferryl intermediates can react with peroxide. These proteins are high-spin in their ferric state with an easily displaced ligand (usually water) at the sixth coordination position. The majority of non-enzymatic electron-transfer haems are low-spin with two strongly bound amino-... 

The combination of photo-Fenton and ozonation results in an important enhancement of the destruction efficiency of organic compounds like phenol [96], 2,4-D [97], aniline or 2,4-chlorophenol ([33] and references therein). As mentioned in Sect. 2.5.1, metal ions catalyze ozone decomposition. In the dark, Fe(II) catalyzes O3 degradation giving the ferryl intermediate (Fe02+, see Sect. 2.6.9), which can directly oxidize the organic pollutant or evolve to a hydroxyl radical ... 

The site in the active Fe ribonucleotide reductase contains two Fe(III) ions 3.3 A apart, bridged by one carboxylate from a glutamate residue and a water-derived oxo bridge (57). The function of this iron center appears to be the formation and stabilization of a free radical on a tyrosine about 5 A away. This radical is formed by reaction of the reduced, diferrous center with 02, probably through peroxide and ferryl intermediates. This unusually stable tyrosyl radical is thought to partic-... 

Y9. Yusa, K., and Shikama, K., Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide Involvement of ferryl intermediate. Biochemistry 26,6684-6688 (1987). 

In chemistry, it is well known that O2 can be strongly bound to a ferrons iron porphyrin in solvents without any protein matrix however, the oxygenated states of most simple iron porphyrins are irreversibly converted into /r-oxodimers (eqnation3), PFe(III)-0-PFe(III), via peroxo and ferryl intermediates (eqnation 2). The /x-oxodimer is usually very stable in solvents, so it is sometimes called a thermodynamic sink. In addition, autoxidation of PFe(II)-02 to an inert ferric porphyrin easily occurs under aerobic conditions (equation 4). Thus, it is clear that the heme pockets of myoglobin and hemoglobin play an important role in protecting the 02-bound heme from dimerization and autoxidation. 

Oxoiron(IV) tefraphenylchlorin complexes have been prepared as the first models of a reaction intermediate in the catalytic cycle of cytochrome d Optical absorption spectra show a characteristic red-shified band at 630 nm as observed in the oxoferryl intermediate of cytochrome d, and the proton NMR spectra of the N-Melm complex exhibit very small hyperfine shifts of the pyrrole protons, as is true for oxoferryl porphyrin complexes. The pyrroline protons of the saturated pyrrole ring show unusual splitting into upheld and downfield resonances. The N-Melm complex also shows normal Fe =0 stretching frequencies as compared to the corresponding oxoferryl porphyrin complexes. And finally, for iron porphycenes, both peroxo and ferryl intermediates have been detected by H NMR spectroscopy during the oxygenation of the Fe complexes. ... 

Figure 6 The most common paradigm for hemoprotein-catalyzed substrate oxidation involves heterolytic scission of the 0-0 bond of an iron-bound peroxo species to give an Fe(IV) = O ferryl intermediate and either a porphyrin radical or a protein radical. The peroxo intermediate is generated in the cytochromes P450 by in situ NAD(P)H-dependent reduction of O2, and in the peroxidases by reaction with H2O2.

---