Free flavin

Synthetic electron acceptors have been shown to react very rapidly with free flavins The combination of a flavin with an apoenzyme often inhibits the reaction with certain electron acceptors. The reaction with an electron acceptor will be thermodynamically favored if its standard reduction potential is larger than that of the flavin. A list of electron acceptors along with their reduction potentials can be foimd in Table 3. 

A.M. Hartnett, C.M. Ingersoll, G.A. Baker, and F.V. Bright, Kinetics and thermodynamics of free flavins and the flavin-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol-gel-derived glass. Anal. Chem. 71, 1215-1224 (1999). 

In the following the basic chemical and physical properties of free flavin will be described in some detail, because the knowledge of these properties is the key for a detailed understanding of the function of flavoproteins. In addition, some general and common properties of the classes of flavoproteins will be presented and discussed in relation to some new concepts. It will however not be possible to cover the whole literature. For the reader interested in more details, the recent review papers by Massey and Hemmerich Bruice , Walsh Simondson and Tollin , and Hemmerich (and references therein) should be consulted. An overview of the pertinent research on flavins and flavoproteins is easily accessible by the proceedings of the international symposia... 

Flavohydroquinone bound to apoproteins plays a very important role in flavo-protein-catalysis, either in the electron-transfer to substrates or other enzymes or in the oxygen activation reaction. The chemical reactivity of 1,5-dihydroflavin bound to apoproteins can differ drastically from that of free flavin. The reactivity is likely governed by factors such as the conformation of the bound flavohydroquinone and the ionization state (cf. below). 

The absorption spectral properties of the neutral and anionic forms are quite different as shown in Fig. 1. Due to the rapid dismutation of flavin radicals to form an equilibrium mixture with the hydroquinone and oxidized forms of the flavin, special procedures must be employed to measure the spectral properties of free flavin radicals. Nearly quantitative amounts of anion radical can be formed in aprotic solvents under basic conditions Alkylation of the N(5) position of the flavin hydroquinone followed by oxidation results in nearly quantitative formation of the... 

The PPy-films doped with (1) were characterized by cyclic voltammetry (Figure 6) and UV/VIS-spectroscopy (Figure 7). The immobilized polymer-bound flavin moieties showed very good electrochemical activity with El4= -0.496 V (vs SCE), which is a little more negative than values (-0.45 V) found in the literature for free flavins (34). If we compare the UV/VIS-spectra (on ITO-electrodes, Figure 7) of... 

Up to 4 kbar, protein is not denatured. However, slight structural changes can be observed, inducing flavin release from its binding site. Free flavin in solution has almost no interactions with the protein, and so energy transfer between the flavin and the amino acids of its binding site decreases, which induces an increase in the fluorescence intensity of the co-factor. 

The motions of free flavin in solution are much more important than those of bound flavin. Thus, the anisotropy of flavin will decrease with increasing hydrostatic pressure. 

Pro-oxidant reactions, reactants Pro-oxidants — Fe+. Cu+ , free flavins and hemes, light Anti-oxidant reactions, reactants... 

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). 

Nagy, J., Ketmey, W. C., and Singer, T. P., 1979, The reaction of phenylhydrazine with trimethylamine dehydrogenase and with free flavins, J. Biol. Chem. 254 268492688. 

The structures of EX and El were deduced by resolution of El into apoenzyme and free flavin-substrate adduct. The structure of this adduct was determined as 5-cyanoethy 1-1,5-dihydro FAD and that of EX was deduced to be a cationic imine resulting from elimination of NO2" from the initial 5-nitroethy 1-1,5-dihydro FAD adduct formed in the process controlled by k2 by nucleophilic attack of nitroethane carbanion on the position of oxidized flavin. The chemistry of flavin reduction by nitroethane carbanion at the active site of D-amino acid oxidase is given by the following scheme (Equation 19) in which the kinetically important... 

The isoalloxazine moiety of the flavin cofactor forms the catalytic heart of a flavoenzyme. It can undergo one- and two-electron redox transitions and form covalent adducts with substrates and protein residues. The redox properties of the flavin cofactor are modulated by the protein environment. In free flavin, the one-electron reduced state is thermodynamically unstable. Elavoenzymes, however, can stabilize the neutral or anionic semiquinone state (Fig. Ic) (44), and they can pass the electrons one at a time to other redox centers. 

Although in some cases an energy-transfer step may be involved, the specific emitter in a bioluminescent reaction is generally an intermediate or product whose excited state is populated during the reaction. Free flavin in aqueous solution has a fluorescence emission maximum at 525 nm, while bacterial luminescence both in vivo and in vitro emits at a maximum around 495 nm. These results and their implications are reported and discussed elsewhere (Balny and Hastings, 1975). 

Holt, R.E. and Cotton, T.M. (1987) Free flavin interference in surface enhanced resonance Raman-spectroscopy of glucose oxidase. Journal of the American Chemical Society, 109,1841-1845. 

Reduction of the radical first in reaction (5) is consistent with the proposed electron-transfer pathway between the hemes in the CCP-yeast cyt c complex (Fig. 16). However, flash photolysis experiments involving rapid bimolecular reduction of native cyts c by free flavin semiquinones (FH ) gave different results (111). The reactions monitored were the following ... 

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