Absorption spectra flavins

Foster Tony Cashmore has consistently argued that we simply can t know what the absorption spectra of the cryptochromes will be. Others have argued differently. The empirical evidence of a comparison between a flavin absorption spectrum and that of the new CRYl over-expression by Ahmad and colleagues suggests that there is a close correlation. 

In contrast to class I flavin reductases, class II flavin reductases are bona fide flavoproteins producing a characteristic flavin-absorption spectrum and containing a bound flavin prosthetic group involved in the electron transfer from NAD(P)H to the flavin substrate. The reaction sequence for class II enzymes, as exemplified by the flavoprotein component of sulfite reductase from E. coli, follows a ping pong bisubstrate-biproduct mechanism (85). In this process, the NAD(P)H and flavin bind to the same site sequentially electrons are first transferred from NADPH to FAD and then from FAD to another flavin. 

D. gigas AOR was the first Mo-pterin-containing protein whose 3D structure was solved. From D. desulfuricans, a homologous AOR (MOD) was purified, characterized, and crystallized. Both proteins are homodimers with-100 kDa subunits and contain one Mo-pterin site (MCD-cofactor) and two [2Fe-2S] clusters. Flavin moieties are not found. The visible absorption spectrum of the proteins (absorption wavelengths, extinction coefficients, and optical ratios at characteristic wavelengths) are similar to those observed for the deflavo-forms of... 

The defenders of the carotenoid-photoreceptor-hypothesis have always understood the shape of these action spectra in the blue to mean that the bluelight receptor is a carotenoid. Indeed, in Fig. 6 3 it can be observed, that the three-peak absorption spectrum of trans-0-carotenoid (in hexene) agrees well with the observed action spectrum of the avena coleoptile (Fig. 3 5). However, there remains one loose end which has been the crucial point of controversy in this field, ever since Galston and Baker66 suggested in 1949 that the photoreceptor for phototropism might be a flavin Flavin absorbs in the near UV, /3-carotenoid does not. 

Fig. 22. (A) Comparison of flavin triplet -> triplet absorption spectra (downwards drawn) with bluelight-induced (440 nm) phototropic curvature of aVena coleoptiles as inhibited by strong monochromatic light in the long wave visible region 154). (B) Comparison of the growth response of Phycomyces induced by strong laser light of wavelength longer than 590nm46, with the flavin phosphorescence spectrum los)...

Fig. 22k. The reasonably good fit between the measured points and the triplet-triplet (T i -> Tn) absorption spectrum of flavin (dotted lines, 174>117)) again suggests a flavin photoreceptor. These authorsIS4) assume an effective decrease of the lifetime of the lowest triplet state by quick triplet-triplet (Tj Tn) turnovers, thereby inhibiting photochemistry (i.e. initiation of phototropism).

The low temperature absorption spectrum of isotropically dissolved flavin resembles blue-light action spectra, that of carotene does not (Fig. 8,49,169)). 

Shinkai and Kunitake (1977b) prepared a hydrophobic flavin analogue, 3-hexadecyl-10-butylisoalloxazine [56]. Its absorption spectrum in CTAB micelles showed distinct shoulders at 420 nm and 460-470 nm, as in the flavin spectrum in organic solvents. This indicates that [56] is located in the hydrophobic region of the micelle. Isoalloxazine [56] bound to a cationic micelle readily oxidizes 2-mercaptoethanol, 1,4-butanedithiol, and thiophenol (Shinkai and Kunitake, 1977b Shinkai et al., 1977a). In non-micellar... 

The visible absorption spectrum of oxidised cellobiose oxidase is typical of cytochrome b (Fig. 5-15). The flavin in cellobiose oxidase is weakly fluorescent, with emission maxima at 564 nm and excitation maxima at 380 and 444 nm. There are no obvious transient changes on reduction that can be readily ascribed to flavin semiquinone, but the strong absorbance of the cytochrome would make such changes difficult to detect. 

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. 

L-Amino acid oxidase is a flavoenzyme that catalyzes the oxidative deamination of L-amino adds. L-Amino acid oxidase activities have been detected in mammals, birds, reptiles, invertebrates, molds, and bacteria [54]. L-Amino acid oxidases show the typical absorption spectrum due to the presence of a molecule of non-covalently bound FAD per subunit (with maxima at 465 and 380nm) they behave like flavoprotein oxidases, as in the case of D-amino acid oxidase. L-Amino add oxidase isolated from rat liver was reported to utilize flavin mononudeotide (FMN) as a co-enzyme, but since it is more active on L-hydroxy acids than on amino adds, it was thus considered as an L-hydroxy add oxidase. Even a partially purified L-amino acid oxidase from turkey Uver appeared to have FMN as a co-factor. 

The resolved complex is composed of two fractions, a soluble part, which comprises about 15% of complex I proteins, and a water-insoluble part consisting of the rest of the protein and the bulk of complex I lipids. The soluble fraction is easily separated from the insoluble material by centrifugation. Upon fractionation with ammonium sulfate, it yields a soluble flavoprotein containing iron and labile sulfide and a dark brown protein, which contains large amounts of iron and labile sulfide but no flavin. The latter appears to be an iron-sulfur protein and exhibits an EPR signal which is characteristic of iron-sulfur center 2 of intact complex I (46). Its absorption spectrum is shown in Fig. 8. The insoluble fraction also contains equimolar amounts of iron and labile sulfide and little or no flavin. 

The absorption band at 384 nm is composed of contributions of the radical species and the second chromophore, whereas the fluorescence spectra with excitation maxima at 398 nm and emission maxima at 470-480 nm are attributed to the pterin alone (146, 155). The 7,8-dihydropterin cofactor, Xmax = 360 nm when free in solution and 390 nm when protein bound, is labile at neutral pH, readily decomposing upon denaturation to form products without significant visible absorption maxima. The photoreduction described above also reduces the second cofactor but in an irreversible manner with complete loss of its fluorescence and visible absorption characteristics (157). Reduction of the blue semiquinone FAD cofactor to the fully reduced form has no effect on the absorption spectrum of the pterin, suggesting that the absorption spectrum of the second cofactor must be independent of the oxidation state of the flavin and that the two cofactors are electronically isolated from each other (157). However, reduction of the flavin radical results in an increase in the fluorescence of the second cofactor, possibly indicating that the flavin radical acts as a potent quencher of fluorescence of the 7,8-dihydropterin. 

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