Neutral flavin semiquinone

Kurfuerst, M., Ghisla, S., and Hastings, J. W. (1986). Bacterial luciferase intermediates the neutral flavin semiquinone, its reaction with superoxide, and the flavin 4a-hydroxide. Method. Enzymol. 133 140-149. 

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...

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... 

The dehydrogenase form of the enzyme is converted to the oxidase form by reversible oxidation of cysteine to form a disulfide bridge. The redox potential of the dehydrogenase form of the enzyme is considerably lower than that of the oxidase form, because the protein confers greater stability on the neutral flavin semiquinone radical (Rajagopalan and Johnson, 1992 Kiskeretal., 1997 Nishino and Okamoto, 2000). 

Inasmuch as the FAD cofactor is capable of accepting two electrons in one-electron steps, with the intermediate formation of a neutral flavin semiquinone, reduction of NADP+ proceeds via two Fd FNR ET reactions. The one-electron reduction potentials for the proteins are as follows [49]. For FNR the values are -331 and —314 mV for the first and second reduction steps, respectively, and for Fd the value is -384 mV. Thus, both ET processes are slightly exoenergetic. Interestingly, complex formation between the two oxidized proteins increases the redox potential difference for the first ET reaction by about 30 mV, thereby increasing the thermodynamic driving force [49]. 

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). 

When PDR is titrated with NADH or dithionite, two stages of reduction are observed. First, the iron-sulfur center and FMN react simultaneously to form a reduced iron—sulfur center and a neutral flavin semiquinone. Further titration reduces the semiquinone to the hydroquinone. During the reduction, a maximum semiquinone concentration of 80% of the total enzyme concentration is reached. The redox potential of the [2Fe-2S] center and that of the oxidized flavin—semiquinone couple are the same, —174 mV. The semiquinone— hydroquinone couple is well resolved from this at —287 mV." These midpoint potentials favor spontaneous electron transfer from NADH to FMN to [2Fe-2S]. 

The proposed mechanism of phosphate elimination in CS involves radicals. After binding reduced flavin and EPSP, a single electron is transferred from reduced flavin to the substrate double bond allowing the elimination of phosphate (Scheme 30). An active site histidine is proposed to protonate the leaving phosphate. Mutagenesis has shown the importance of this residue for catalysis. " The neutral flavin semiquinone then oxidizes the intermediate substrate radical. The structure of the ternary complex with flavin and EPSP shows that N5 of the flavin is directly under C6 of the substrate, implying a direct role of N5 in the reaction. ... 

Radical flavin species have not been detected during turnover of CS therefore, these species must be short lived. However, a radical flavin species was observed using the EPSP analogue (6R)-6-fluoro-EPSP. Phosphate is eliminated from this analogue however, due to the replacement of the C6 pro-R hydrogen with fluorine, the reaction cannot proceed further, resulting in the formation of a substrate-derived radical and neutral flavin semiquinone. 

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

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

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

Fig. 4. Simulated ESR spectrum of a flavin semiquinone considering only the hyperfine interactions from the strongly coupled (N(5) and N(10) nitrogens (left) and and experimentally observed ESR spectrum of a deuterated flavodoxin neutral semiquinone in HjO (right)...

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. 

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. 

Fio. 3. (a) Typical neutral (blue) flavin semiquinone produced upon anaerobic... 

The redox states of the flavin cofactor in a purified flavoenzyme can be conveniently studied by optical spectroscopy (see also Elavoprotein Protocols article). Oxidized (yellow) flavin has characteristic absorption maxima around 375 and 450 nm (Fig. lb and Ic). The anionic (red) and neutral (blue) semiquinone show typical absorption maxima around 370 nm and 580 nm, respectively (Fig. lb and Ic). During two-electron reduction to the (anionic) hydroquinone state, the flavin turns pale, and the absorption at 450 nm almost completely disappears (Fig. lb and Ic). The optical properties of the flavin can be influenced through the binding of ligands (substrates, coenzymes, inhibitors) or the interaction with certain amino acid residues. In many cases, these interactions result in so-called charge-transfer complexes that give the protein a peculiar color. 

Several properties of these enzymes are shared irrespective of the identity of the flavin (FMN or FAD). Many react with sulfite to form N5 flavin adducts, which is readily detectable spectroscopically. In addition, they stabilize the benzoquinoid resonance structure of 8-mercaptoflavin when it is substituted for the namral prosthetic group, producing a characteristic intense absorbance band at 600 nm. They also stabilize semi-quinone. Since the pA), of the stabilized semiquinone is often low, the anionic rather than the neutral (blue) semiquinone is detected. However, there are a few cases where the pA), is experimentally accessible and both anionic and neutral semiquinones are observed. These general features are the consequence of a positive charge (contributed by either a protein residue or the positive end of a helix dipole) at the Nl—C2=0 locus of the flavin. 

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...

Gallagher and co-workers have characterized the reductase component by EPR and fluorescence spectroscopy. They showed that it contained one FAD and a [IFe-lS] " cluster. The FAD could be reduced in a two-step reaction to the fully reduced flavin. The optical spectrum of the semiquinone species was typical of a neutral flavin radical. The [2Fe-2S] + cluster could also be reduced by one electron equivalent to [2Fe-2S] +. Both paramagnetic species could be detected by EPR. It could also be shown by a combination of mid-point potential measurements and electron-transfer kinetics that this component could supply the energy required for the epoxygenation reaction. 

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. 

A comprehensive series of oxidation-reduction potential measurements have shown the FAD moiety to have the following one-electron couples PFl/PFIH = = —290 mV and PFIH 7PFIH2 = —365 mV while the FMN moiety exhibits the following PFl/PFl- = -110 mV and PFIH /PFIH = -270 mV. The FMN and FAD smiquinones were found to both be the neutral form as judged from absorption and ESR spectral data. The overlap of oxidized/semiquinone potential of the FAD moiety withkhe semiquinone/hydroquinone couple of the FMN moiety demonstrates the thermodynamic facilitation of flavin-flavin electron transfer via a one-electron mechanism. Stopped-flow kinetic data are also consistent with this view in... 

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