Half-Reduced Flavins

A failure by one of us to take fully into account the presence of inactivated xanthine oxidase, leading to misinterpretation of incomplete reaction of enzyme with iodoacetamide and hence to the apparently erroneous conclusion, that the two FAD molecules in the enzyme were non-equivalent (72), may serve as a warning to others. This reagent has since been shown to alkylate the flavin of reduced xanthine oxidase molecules, whether these are of the active or inactivated forms (73). Thus, under conditions where little of the inactivated form is reduced, the reagent becomes a specific one for the active enzyme (20). In the original experiments (59, 72) the content of active enzyme was, by coincidence, rather close to half of the total enzyme present. Thus, the presence of inactivated enzyme, rather than a lack of reactivity of one... 

Figure 5.3 The flavin coenzymes FAD and FMN. Note that in contrast to NAD+, flavins can be half-reduced to the stable radical FADH or fully reduced to the dihydroflavin shown.

The addition of one half equivalent of e.g. dithionite to a solution of (12) yields a so-called half-reduced system. Half-reduced is not a very meaningful term, it only implies that one electron is added to flavin without any indication of the spin distribution. This is best demonstrated by equilibrium (1)... 

The metal chelating ability of flavins has been extensively investigated predominately in Hemmerich s laboratory While the oxidized and reduced (hydroquinone) forms of flavin are poor chelating ligands to divalent metal ions in aqueous solution, the flavin semiquinone forms chelates of considerable stability. This is readily demonstrated by the increase in semiquinone formation on the addition of metal ions to a half-reduced flavin solution which occurs by shifting the equilibrium away from semiquinone dismutation ... 

Figure 15-13 Properties of oxidized, half-reduced, and fully reduced flavins. See Muller et al.263 26i...

PQQ and the other quinone prosthetic groups described here all function in reactions that would be possible for pyridine nucleotide or flavin coenzymes. All of them, like the flavins, can exist in oxidized, half-reduced semiquinone and fully reduced dihydro forms. The questions to be asked are the same as we asked for flavins. How do the substrates react How is the reduced cofactor reoxidized In nonenzymatic reactions alcohols, amines, and enolate anions all add at C-5 of PQQ to give adducts such as that shown for methanol in Eq. 15-51, step a 444,449,449a Although many additional reactions are possible, this addition is a reasonable first step in the mechanism shown in Eq. 15-51. An enzymatic base could remove a proton as is indicated in step b to give PQQH2. The pathway for reoxidation (step c) might involve a cytochrome b, cytochrome c, or bound ubiquinone.445 446... 

The spectra of oxidized glutathione reductase and of the 2-electron-reduced enzyme are shown in Fig. 1. This red intermediate, which has been shown to be functional in catalysis 39), will be referred to as EH2 designating a half-reduced active center it has also been referred to as F (S3, 53), but this can be confused with oxidized flavin in other nomenclatures. Its spectral characteristics are virtually identical with those of the analogous species of lipoamide dehydrogenase 34, 37, 64)- It has... 

The catalytic cycle of each flavoenzyme consists of two distinct processes, the acceptance of redox equivalents from a substrate and the transfer of these equivalents to an acceptor. Accordingly, the catalyzed reactions consist of two half-reactions a reductive half-reaction in which the flavin is reduced and an oxidative half-reaction, in which the reduced flavin is reoxidized. This review summarizes the chemistry of simple flavoprotein reductases, dehydrogenases, (di)thiol oxidoreductases, oxidases, and monooxygenases (Table 1) (5 0) This grouping provides a good appreciation about what type of common mechanisms can be distinguished and what type of substrates can be converted. Information on the chemistry of complex flavoenzymes can be found in the Further Reading section. 

The catalytic cycle of each flavoenzyme consists of a reductive half-reaction, in which the flavin is reduced, and an oxidative half-reaction, in which the reduced flavin is reoxidized. The reduction and oxidation steps are in many cases irreversible, which enables the direct characterization of reaction intermediates (see See Also section and the Further Reading List). 

Figure 7.10 (a) Structure of flavin and of FAD. (b) Oxidative half-reaction of flavin enzymes, (c) Half-reduced flavin in two ionisation states os... 

Flavin catalysis may involve three oxidation levels of the ring, and each of these may exist as a cation, anion or neutral compound, giving a total of 9 forms. The oxidation levels are flavoquinone (oxidized) flavosemiquinone (half-reduced) flavohydroqui-none (reduced). Some catalytic mechanisms involve only one electron, i.e. the flavoquinone is converted to the semiquinone, or the semiquinone is converted to the hydroquinone. Other mechanisms involve two electrons, and the flavin shuttles back and forth between the quinone and the hydroquinone states. FAD is biosynthesized from flavin mononucleotide by the action of FAD pyrophosphatase (EC 3.6.1.8) FMN + ATPseFAD + PP,. 

A similarly wide-spread abuse is the confusion of half reduced and flavin radical . The term half reduced does not imply any information about spin concentration in a flavin system, as the electrons might be... 

As mentioned above, Beinert 3) was first to see flavin radicals in neutral solution, while Michaelis and Schwarzenbach 129) could only infer their existence in the largely disproportionated state of the half reduced neutral flavin system. There are several means for shifting equilibrium between oxidized and reduced flavin towards the radical side ... 

Deprotonation Since blue HFl ( = 5-HFl, pK 8.5, cf above) is more acidic than Flox (pK lO) (for chemical studies this dissociation should in any case be blocked by N(3)-prealkylation), alkalinization of the half-reduced free flavin system will again enhance radical formation (70) according to ... 

Independent support for interflavin o-contacts comes from recent chemical studies by Favaudon and Lhoste (13,14). The french authors describe, as already anticipated, a nearby diffusion-controlled dimer formation in aprotic polar medium as the first step in the interflavin contact between oxidized and reduced states, which would finally yield two flavin radicals. This dimer was shown to be not identical in any respect with the well known quinhydrone which can only be obtained in aqueous systems at high flavin concentrations. The long wave band in the absorption spectrum of the new dimer appears to be of charge transfer type, but with a highly reduced half width and better resolved shape than the flavoquinhydrone spectrum. 

The lack of reactivity of the semiquinone per se with either thioredoxin or NADPH shows that it cannot be involved in catalysis. The rapid production of semiquinone by irradiation of partially reduced enzyme is a light-activated disproportionation since it is totally dependent upon the presence of some oxidized enzyme. Enzyme fully reduced by dithionite forms no semiquinone, while enzyme partially reduced by dithionite rapidly forms semiquinone upon irradiation. Furthermore, the light-activated disproportionation of enzyme first reduced with NADPH results in the reduction of NADP. Thus, FAD catalyzes the disproportionation in keeping with the known photosensitizing nature of free flavins. This reaction is reversed slowly (half-time ca. 150 min 25°) in the dark. The semiquinone is rapidly reoxidized by oxygen to yield an enzyme with unaltered spectral and catalytic properties (58). Similar reactions have been very briefly reported for lipoamide dehydrogenase the dark reverse reaction is comparatively rapid, being complete in 30 min (16S). 

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. 

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