Substrate radicals

Substrates Radical and Source" o Isomer (%) m p Reactivitv ffiK) ... 

Evidence in support of radical intermediates with MMO from both M. capsulatus (Bath) and M. trichosporium 0B3b was reported from experiments in which substrate radicals were trapped during turnover (89, 90). The amount of trapped radical, however, was not quantified in these experiments. In other reports, no diffusable radical species were detected in reactions with MMO from M. trichosporium 0B3b (61). 

In summary, mechanistic studies have revealed intriguing differences between MMO from M. capsulatus (Bath) and MMO from M. triehosporium OB3b. With M. capsulatus (Bath), radical clock substrate probes indicated either that a substrate radical is not produced or that it reacts with a rate constant > 1013 s-1. With MMO from M. triehosporium OB3b, radical involvement was suggested from several experiments, and a rebound rate constant of 6 x 1012 s"1 was calculated for this system. 

We have not so far mentioned the Phase III increase in the Rapid signal (Fig. 5). It seems (67) that Phase II represents over reduction of molybdenum to Mo(IV), possibly by substrate radicals (see Section V H). The system then comes towards thermodynamic equilibrium by interaction between reduced active enzyme molecules and oxidized inactive ones (67, cf. 64). As Mo(IV) of the former is oxidized to Mo(V), during Phase III, so iron or flavin of the inactive enzyme is reduced. Later, in Phase IV, molybdenum of the inactive enzyme is reduced also to give the Slow signed. Alloxanthine, which as noted above, forms a stable complex with Mo(IV), seems to abolish both the slow phase in the 450 nm bleaching of the enzyme by xanthine and the Phase III increase in Rapid signal (91). 

In accord with this mechanism, free peroxyl radical of the reaction product hydroperoxide activates the inactive ferrous form of enzyme (Reaction (1)). Then, active ferric enzyme oxidizes substrate to form a bound substrate radical, which reacts with dioxygen (Reaction (4)). The bound peroxyl radical may again oxidize ferrous enzyme, completing redox cycling, or dissociate and abstract a hydrogen atom from substrate (Reaction (6)). 

Scheme 2. Simplified scheme for oxidative N-dealkylation incorporating a cytochrome P-450 perferryl oxygen intermediate and substrate radicals.

An additional effect of the cosensitization may lead to different products or product ratios, which is caused by the efficient sensitizer radical ion—substrate ion separation. This separation inhibits the early back-electron transfer to the substrate radical ion or early intermediates and favors products of complex reaction pathways (late ETc)... 

The mechanism of cytochrome P450 catalysis is probably constant across the system. It is determined by the ability of a high valent formal (FeO) species to carry out one-electron oxidations through the abstraction of hydrogen atoms or electrons. The resultant substrate radical can then recombine with the newly created hydroxyl radical (oxygen rebound) to form the oxidized metabolite. Where a heteroatom is the (rich) source of the electron more than one product is possible. There can be direct recombination to yield the heteroatom oxide or radical relocalization within the... 

In eukaryotes, ribonucleotide reductase is a tetramer consisting of two R1 and two R2 subunits. In addition to the disulfide bond mentioned, a tyrosine radical in the enzyme also participates in the reaction (2). It initially produces a substrate radical (3). This cleaves a water molecule and thereby becomes radical cation. Finally, the deoxyribose residue is produced by reduction, and the tyrosine radical is regenerated. 

Peroxydisulphate ions are reduced by solvated electrons to give tbe sulphate radical-anion which is a powerful oxidising agent, functioning by single electron transfer to form sulphate dianion, fhus irradiation of solutions containing peroxydisulphate and an alkyl or alkoxybenzene gives the substrate radical-cation in a diffusion-controlled reaction [63],... 

The relationships between rate of cleavage, bond strength and radical-anion redox potential can be combined in one concept. In this, cleavage rate is dependent on a reaction driving force, defined as the difference between the redox potential of the substrate radical-anion and the redox potential of the product anion in equ-librium with the coiresponding radical (E° for bromine ion, bromine radical as an example). 

Methylbenzenes lose a proton from a methyl group to form a benzyl radical. In aqueous M-percbloric acid this reaction is fast with a rate constant in the range 10 lO s and the process is not reversible [24]. The process becomes slower as the number of methyl substituents increases, Hexaethylbenzene radical cation is relatively stable. When the benzyl radical is formed, further reactions lead to the development of a complex esr spectrum. Anodic oxidation of hexamethylbenzene in trifluoroacetic acid at concentrations greater than 1 O M yields the radical-cation I by the process shown in Scheme 6.1 [14], Preparative scale, anodic oxidation of methylbenzenes leads to the benzyl carbonium ion by oxidation of the benzyl radicals formed from the substrate radical-cation. Products isolated result from further reactions of this carbonium ion. 

As exemplified in Figure 2, Type 1 mechanism, electron transfer from L to sens yields two radicals, the substrate radical, L", and the sensitizer radical anion (sens ). In the next step, the lipid radical may induce a chain peroxidation cascade involving propagation reactions -The sensitizer radical anion may also start a sequential one-electron reduction of 2 generating HO in the presence of reduced transition metals. As a result, this may lead to abstraction of a lipid allylic hydrogen with subsequent generation of a carbon-centered lipid radical, L, that is rapidly oxidized to a peroxyl radical (vide supra). 

With ions or dipolar substrates, radical ions undergo nucleophilic or electrophilic capture. Nucleophilic capture is a general reaction for many alkene and strained-ring radical cations and may completely suppress (unimolecular) rearrangements or dimer formation. The regio- and stereochemistry of these additions are of major interest. The experimental evidence supports several guiding principles. 

Inasmuch as flavins can accommodate two electrons but possess a relatively stable one-electron intermediate, an obvious question which can be asked of any flavin-mediated two electron redox reaction is whether or not the mechanism includes the radical species on a direct line between reactants and products. The mere observation of semiquinones in a reaction mixture is not sufficient evidence for their intermediacy, due to the existence of side reactions such as comproportionation (F -I- FH2 2 FH-) which can generate radicals rapidly. Bruice has discussed this question from a physical-organic point of view and concluded that there must exist a competition between one-electron and two-electron processes and that the actual mechanism should be determined mainly by the free energy of formation of substrate radical and the nucleophilicity of the substrate. Bruice has analyzed a variety of systems which he feels should proceed via one-electron mechanisms among these are quinone and carbonyl group reduction by FH2... 

A long-known characteristic of D-amino acid oxidase is its tendency to form charge-transfer complexes with amines, complexes in which a nonbonding electron has been transferred partially to the flavin. Complete electron transfer would yield a flavin radical and a substrate radical which could be intermediates in a free radical mechanism, as discussed in the next section.256... 

Another important experiment398 showed that lsO from [2-lsO]propanediol was transferred into the 1 position without exchange with solvent. Furthermore, lsO from (S)-[l-lsO]propanediol was retained in the product while that from the (R) isomer was not. Thus, it appears that the enzyme stereospecifically dehydrates the final intermediate. From these and other experiments, it was concluded that initially a 5 -deoxyadenosyl radical is formed via Eq. 16-35. This radical then abstracts the hydrogen atom marked by a shaded box in Eq. 16-36 to form a substrate radical and 5 -deoxy-adenosine. One proposal, illustrated in Eq. 16-36, is that the substrate radical immediately recombines with... 

At present most evidence favors, for the isomerization reactions, an enzyme-catalyzed rearrangement of the substrate radical produced initially during formation of the 5 -deoxyadenosine (Eq. 16-38).40CM01c... 

Ordinarily, free-radical chain reactions begin by abstraction of a weakly bonded hydrogen atom (34) from the substrate by a radical which is regenerated in a subsequent step in the process. The resultant substrate radical may then rearrange or undergo decomposition reactions similar to the elimination of ions by consecutive electron displacements (48,49). The path to the non-labeled formic acid begins with abstraction... 

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