Anionic flavin semiquinone

II Properties of Neutral and Anionic Flavin Semiquinones in Model Systems... 

Fig. 1. Absorption spectra of neutral and anionic flavin semiquinones. (-) Anionic riboflavin...

Fig. 3. H-ENDOR spectra of a protein-bound anionic flavin semiquinone (oxynitrilase) and a protein-bound neutral flavin semiquinone (Azotobacter flavodoxin). The ENDOR spectra were recorded at the magnetic field settings indicated. Taken from Ref. with permission...

ESR and ENDOR spectral data on flavin-metal chelates in which the metal has a magnetic moment (e.g. Zn, Cd, Cd) have shown that the hyperfine couplings on the flavin are comparable to those of the anionic flavin semiquinone (as is the... 

Each of the forms of ETF isolated from the different sources contain FAD as coenzyme and form an anionic semiquinone on one-electron reduction. Stopped-flow kinetic studies on the pig liver ETF showed the anionic flavin semiquinone to be formed at times faster than catalytic turnover and thus demonstrate the participation of the anionic FAD semiquinone as an intermediate in the acceptance of reducing equivalents from the dehydrogenase. These studies would also imply the intermediacy of the semiquinone form of the acyl CoA dehydrogenase which would have been expected to form a neutral flavin semiquinone at the time the studies of Hall and Lambeth were performed, however, no spectral evidence for its formation were found. Recent studies have shown that the binding of CoA analogs to the dehydrogenase results in the perturbation of the pKa of the FAD semiquinone such that an anionic (red) rather than the neutral (blue) semiquinone is formed. This perturbation was estimated to reduce the pKa by at least 2.5 units to a value of... 

These data would suggest that both the dehydrogenase and ETF form anionic flavin semiquinone on electron transfer which cannot be differentiated spectrally, and thus the kinetic intermediacy of the anionic flavin semiquinone in electron transfer between the dehydrogenase and ETF may be due to both flavoproteins. 

The properties of the semiquinone from of the ETF isolated from the methylotrophic bacterium resemble those of the bacterial flavodoxins with the exception that flavodoxins form neutral semiquinones whereas this ETF forms an anionic semiquinone. Nearly quantitative anionic semiquinone formation is observed either in the presence of excess dithionite or when excess trimethylamine and a catalytic amount of trimethylamine dehydrogenase are added. Of interest is the apparent stability of the anionic semiquinone towards oxidation by O2 but not to oxidizing agents such as ferricyanide. This appears to be the first example of an air-stable protein-bound anionic flavin semiquinone. Future studies on the factors involved in imparting this resistance to O2 oxidation by the apoprotein are looked forward to with great interest. 

Disaccharides and even some insoluble polysaccharides are substrates, but not monosaccharides. Cellobiose oxidase is unusual among flavoproteins, as it stabilises the red anionic flavin semiquinone and forms a sulphite adduct, yet appears to produce the superoxide anion as its primary reduced oxygen product. 

GALDH shows a high enantio-preference for L-galactono-1,4 -lactone (13). Reoxidation of the two-electron reduced enzyme by cytochrome c occurs in two single-electron steps and involves the intermediate formation of the red anionic flavin semiquinone (Fig. lb). Related aldonolactone oxidoreductases act as true oxidases, which suggests that they provide a better access of molecular oxygen to the active site. 

Two ionic forms of flavin semiquinones have been shown to exist in flavoenzymes the neutral form and the anionic form... 

A considerable amount of information regarding flavin semiquinone reactivity as well as the environment of the bound flavin coenzyme has accumulated over the years from studies of flavoenzyme systems which produce semiquinones either on photochemical reduction or upon reduction by one electron equivalent of dithionite, but which do not form a detectable semiquinone intermediate during catalytic turnover. For example, the correlation of anionic semiquinone formation and the ability to bind sulfite at the N(5) position in a number of flavoenzyme... 

No major differences in rate were observed with the ionic form (anion or neutral) of the flavin semiquinone. Comparison of the rates of reduction by a number of flavin analogs suggests that cytochrome interaction occurs through the N(5)-dimethylbenzene region of the isoalloxazine ring. 

Almost all biological tissues contain some organic free radicals that are detectable by ESR. These radicals ( tissue radicals ) are of low reactivity in the sense that they do not react readily with molecules in the system, in particular oxygen if this is present. Thus, they tend to be radicals at the end of a radical chain. Examples are the ascorbate radical anion, melanin free radicals, and some other oxygen-insensitive species, such as some flavin semiquinones. The magnetic properties of these various radicals are sufficiently distinct that their ESR spectra can be differentiated on the basis of g value and linewidth. 

Free ACADs stabilize the neutral flavin semiquinone when the enzyme is artificially reduced by singleelectron donors however, binding of the product enoyl-CoA esters shifts the pAi, of the semiquinone to 7.3 to generate the anionic form. The two-electron redox potential of free acyl-CoA substrates (—40 mV) is not low enough to produce free reduced enzyme (—145 mV for MCAD). However, ligands bind much more tightly to the reduced enzyme, shifting the redox equilibrium to promote flavin reduction. For example, the dissociation constant of oct-2-enoyl-CoA from oxidized enzyme is 200 nmol 1, but with reduced enzyme it is 13 pmoll . Therefore, it is thermodynamically unfavorable for the reduced enzyme to release product. Instead, turnover occurs by sequential one-electron oxidations by ETF while the product enoyl-CoA ester is bound " (Scheme 11). 

The proposed chemical mechanism starts with generation of the excited state of the reduced flavin, followed by transfer of a single electron to the pyrimidine dimer (Scheme 31). The ketyl radical formed is presumed to decompose in steps to a pyrimidine-pyrimidine anion radical pair, which transfers a single electron to the flavin semiquinone, regenerating reduced flavin. The repaired DNA is then released. " ... 

In studies of paramagnetic flavin species application of EPR has traditionally been very valuable to distinguish the protonation state of flavin semiquinones by means of the signal width of its typical inhomogeneously broadened EPR resonance centered at gi o = 2.0034 [22, 23]. Anion flavin radicals (deprotonated at N5) show peak-to-peak line widths (of the EPR signals in the first derivative) of around... 

Figure 11.9 Flavoenzyme catalyzed electron transfer and oxidation/oxygenation reactions The extensive conjugation of the isoaUoxazine ring system results in the yellow chromophore ( ax = 450 nm) in the oxidized flavin. Flavin semiquinones are stable radicals, because the unpaired electron is highly delocalized through the conjugated isoaUoxazine structure. The neutral semiquinone is blue = 570 nm) and the flavosemiquinone anion is red (A ax = 480 nm). The...

In the presence of an excess of pyruvate, it is possible to poise the enzyme in a redox state such that the FMN group is essentially in the semiquinone state and the heme is either reduced (paramagnetic) or oxidized (diamagnetic). A previous EPR study carried out on cytochrome b2 from Hansemla anomala by Bertrand and co-workers showed that the EPR signals of the anionic flavin radical are identical in both preparations the difference between their linewidths is less than 0.05 mT. By using a simplified model in which the heme was... 

The above considerations provide a rationale for the redox properties of flavodoxin which function between the flavin hydroquinone and neutral semiquinone redox forms. Further studies are required to determine whether similar properties exist in flavoproteins in which both redox couples (PF/PFl- and PFI/PFIH2) are operative and in situations where the anionic semiquinone rather than the neutral form is functional. 

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... 

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