Isoalloxazines radicals

The same conclusions hold for the neutral and anionic isoalloxazine radicals, e.g., 133 and 134.401 The neutral radicals have been studied by Ehrenberg... 

Flavin coenzymes can exist in any of three different redox states. Fully oxidized flavin is converted to a semiqulnone by a one-electron transfer, as shown in Figure 18.22. At physiological pH, the semiqulnone is a neutral radical, blue in color, with a A ax of 570 nm. The semiqulnone possesses a pAl of about 8.4. When it loses a proton at higher pH values, it becomes a radical anion, displaying a red color with a A ax of 490 nm. The semiqulnone radical is particularly stable, owing to extensive delocalization of the unpaired electron across the 77-electron system of the isoalloxazine. A second one-electron transfer converts the semiqulnone to the completely reduced dihydroflavin as shown in Figure 18.22. 

Flavin redox states in a dual flavin enzyme. (Left) Single-electron reduction of the isoalloxazine ring generates the semiquinone radical, while reduction by two electrons generates the fully reduced species. (Right) Five possible oxidation levels of a dual flavin enzyme, where the FMN reduction potential is held at a more positive value relative U) FAD. The flavins can theoretically accept a maximum of four electrons obtained from two NADPH. However, in NADPH-cytochrome P450, reductase, full reduction of the flavins is not normally reached when NADPH serves as the reductant. 

The remainder of this section is devoted to cases in which an immobilized oxidizing reagent can be regenerated by the action of a simple oxidant, such as a peroxide or O2. For instance, the flavin 10-ethyl-isoalloxazine was immobilized on organic polymers and used in the air oxidation of 1 -benzyl-1, 4-dihydronicotinamide (395). However, peracids, dioxiranes, and nitroxyl radicals are of much more synthetic importance. 

The isoalloxazine nucleus of the flavins [3-(R or H)-7,8-dimethyl-lO-R -isoalloxazines] may exist in the fully reduced (1,5-di-hydro-), the radical (semiquinone), and the fully oxidized (quinone) states. Because of acid-base equilibria, each of these oxidation states... 

Radicals from Pteridine, Isoalloxazine, and Related Systems.275... 

It is beyond the scope of this chapter to deal in depth with the complex array of radicals deriving from pteridine and isoalloxazine, in particular, those which are derived from naturally occurring pterin and flavin compounds. Accordingly, coverage is restricted to simpler examples and their elementary properties. 

The vast majority of flavoenzymes catalyze oxidation-reduction reactions in which one substrate becomes oxidized and a second substrate becomes reduced and the isoalloxazine ring of the flavin prosthetic group (Figure 1) serves as a temporary repository for the substrate-derived electrons. The catalytic reaction can be broken conveniently into two steps, a reductive half reaction (from the viewpoint of the flavin) and an oxidative half reaction. The flavin ring has great utility as a redox cofactor since it has the ability to exist as a stable semiquinone radical. Thus, a flavoenzyme can oxidize an organic substrate such as lactate by removal of two electrons and transfer them as a pair to a 2-electron acceptor such as molecular oxygen, or individually to a 1-electron acceptor such as a cytochrome. 

Fenner and co-workers have described radicals in which an NH from the pyrazine ring of lumazine or isoalloxazine derived radicals is replaced by S (e.g., 273-275) (cf. Part I Section III,C,5). Cation-radicals were generated by solution of the parent heterocycles in concentrated sulfuric acid or by oxidation by dibenzoyl peroxide in trifluoracetic acid-containing... 

G [139], These widths correlate with the optical spectra of the radicals semiquin-ones with a 19 G linewidth have pronounced absorption between 550 and 650 nm and appear blue those with a 15 G linewidth (Fig. 8a) have spectra with peaks between 485 and 370 nm and appear red. The additional width of the 19 G spectrum is due to an exchangeable proton it has been shown that the linewidth decreases to 15 G in DjO solutions. Experiments on model compounds [141] indicate that the blue type of radical is a neutral semiquinone with the proton on N(5) of the isoalloxazine ring (9), and that the red species is either the semiquinone anion or the neutral 0(4)-enol tautomer. Covalently bound semiquinones have ESR spectra that are distinctly narrowed [142-144], having a width of around 12 G (Fig. 4b). The reason for this is that the covalently bound flavins lack a methyl group at C-8, which when present makes a significant contribution to the total linewidth. 

The transient absorption spectra similar to that of the ion-pair state of indole cation radical and flavin anion radical were also observed in D-amino acid oxidase (5), although the spectra were not so clear as those of flavodoxin. In D-amino acid oxidase, the coenzyme, flavin adenine dinucleotide (FAD), is wealtly fluorescent. The fluorescence lifetime was reported to be 40 ps (16), which became drastically shorter (less than 5 ps) when benzoate, a competitive inhibitor, was combined with the enzyme at FAD binding site (17). The dissociation constant of FAD was also marlcedly decreased by the binding of benzoate (17). These results suggest that interaction between isoalloxazine and the quencher became stronger as the inhibitor combined with the enzyme. Absorbance of the transient spectra of D-amino acid oxidase-benzoate complex was remarkably decreased. In this case both rate constants of formation and decay of the CT state could become much faster than those in the case of D-amino acid oxidase free from benzoate. 

Obviously, more work is required to further substantiate the presence of the proposed radical intermediates in the p-hydroxybenzoate hydroxylase reaction, possibly via EPR and spin-trapping studies. Studies by Detmer and Massey 247) on phenol hydroxylase have indicated that the reaction rate constants for the conversion of meta-substituted substrates plotted versus the Hammett parameters yield a straight line of slope equal to 0.5. This is consistent with the mechanism proposed by Anderson, as the negative slope is expected for an electrophilic aromatic substitution reaction, while the small magnitude of the slope may be indicative of a radical mechanism. Furthermore, recent work by Massey and co-workers on p-hydroxybenzoate hydroxylase utilizing 6-hydroxy-FAD as cofactor and p-aminobenzoate as substrate indicated that the absorption spectrum of intermediate 67 exhibited a satellite band at 440 nm 248). Anderson et al. suggest that the satellite band may result from the formation of an aromatic phenoxyl radical at the C-6 position of the isoalloxazine ring of the flavin 244). This species would result from a shift of the initial peroxyl radical center from C(4a) to C-6 via N(5) 245). 

It is thought that the mechanism involves abstraction of the proton a to the carboxyl group followed by oxidation of the resulting carbanion. The exact nature of the oxidation step - 1-electron vs. 2-electron transfer and the importance of flavin N5 or 43 adducts - is presently unknown. Nucleophilic addition can take place by hydride or carbanion attack at N5 or 4a of the isoalloxazine (Scheme 5). However, there is radical trapping and CIDNP evidence that carbanion oxidation can take place by 1-electron transfer. 1-Electron transfer from a carbanion to the electron-deficient, but aromatic, oxidised flavin has been observed in model systems (Scheme 5). 

So far, no single-crystal EPR studies of flavin radicals have been reported. However, the orientations of the g-principal axes relative to the molecular frame of the flavin s isoalloxazine moiety have been derived from orientation-selection effects of the quite anisotropic hyperfine coupling of H5 (or D5 in an isotope-exchange experiment) of the neutral flavin radical, both with EPR [28] and ENDOR... 

As expected, the Z-principal axis of g is oriented perpendicular to the 71-plane of the flavin ring. Analyses of EPR and ENDOR data revealed angles of (—29 4)° and (—14 2)° between the X axis of g and the N5-H5 (or N5-D5) bond in (6 ) photolyase [38] and cyclobutane pyrimidine dimer (CPD) photolyase [28, 30], respectively. The factors that cause the reorientation of the X and Y axes of g of a neutral flavin radical in the two highly homologous cofactor binding pockets of CPD photolyase and (6 ) photolyase remain elusive. Also, the orientations of the principal axes of g relative to the molecular frame of the isoalloxazine ring of a flavin anion radical still need to be determined experimentally. 

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