Flavoproteins chromophores

Foster Yes I agree, there has been much discussion about the absorption spectrum of the cr3rptochromes because of the two potential chromophores, but what is fascinating is that the normalized flavoprotein and CRY 1 action spectrum I showed in my presentation (see Fig. 4) shows a striking similarity with that action spectrum iot Arabidopsis CRYl as published by Margaret Ahmad and colleagues (Ahmad et al 2002). 

Flavin-containing enzymes are known as flavoproteins and, when purified, normally contain their full complements of FAD or FMN. The bright yellow color of flavoproteins is due to the isoalloxazine chromophore in its oxidized form. In a few flavoproteins, the coenzyme is known to be covalently bonded to the protein by means of a sulfhydryl or imidazole group at the C-8 methyl group and in at least one case at C-6. In most flavoproteins, the coenzymes are tightly but noncovalently bound, and many can be resolved into apoenzymes that can be reconstituted to holoenzymes by readdition of FAD or FMN. 

The spectra of adrenodoxin and testis iron protein reduced either by dithionite or by NADPH plus adrenodoxin reductase (flavoprotein) exhibit a distinct feature (Fig. 2) the absorption maxima at 455 mp, 414 mp, and 320 mp graetly decrease, although they do not disappear completely. A new distinct maximum appears at 540 mp. However, adrenodoxin can not be reduced by ascorbate or borohydride. The chemical reduction by an excess amount of dithionite gives an 86% decrease in the absorbance, whereas the enzymatic reduction produces a 65% decrease. This difference can be interpreted as gradual loss of iron from the chromophore upon reduction by dithionite. When the reduced form is reoxidized by air, the original spectrum can be regained. The identical characteristic is observed in preparations of testis iron protein. Therefore, adrenal and testis non-heme iron proteins are autoxidizable as well as bacterial and spinach ferredoxins are. 

In this chapter results of the picosecond laser photolysis and transient spectral studies on the photoinduced electron transfer between tryptophan or tyrosine and flavins and the relaxation of the produced ion pair state in some flavoproteins are discussed. Moreover, the dynamics of quenching of tryptophan fluorescence in proteins is discussed on the basis of the equations derived by the present authors talcing into account the internal rotation of excited tryptophan which is undergoing the charge transfer interaction with a nearby quencher or energy transfer to an acceptor in proteins. The results of such studies could also help to understand primary processes of the biological photosynthetic reactions and photoreceptors, since both the photoinduced electron transfer and energy transfer phenomena between chromophores of proteins play essential roles in these systems. 

The enzyme D-lactate dehydrogenase from Megasphaera elsdenii catalyzes the oxidation of D-lactate to pyruvate, with an electron-transferring flavoprotein serving as the ultimate oxidant. Its reaction is similar to the first step of the lactate oxidase reaction, but the two enzymes use enantiomeric substrates, leading to the proposal that the two enzymes utilize similar mechanisms but bind their substrates in opposite orientations (Ghisla et al., 1976). Incubation of o-lactate dehydrogenase with d-13 leads to enzyme inactivation with a partition ratio of 5 (Olson et al., 1979). A novel pink chromophore formed concomitantly... 

Protein fluorescence is also a useful signal in such studies. In some cases, in addition to its aromatic amino acid residues, an enzyme may possess other useful chromophores. Thus rapid reaction studies of haemoproteins and flavoproteins, for example, have relied heavily on the spectral properties of the prosthetic groups of these enzymes. 

Proteins that have tightly bound cofactors, such as heme proteins, photosynthetic reaction centers and antenna proteins, flavoproteins, and pyridoxal phosphate- and NAD-dependent enzymes, provide a variety of chromophores which have absorption bands in the visible and UV region. The CD bands associated with the chromophoric groups are frequently quite intense, despite the fact that the isolated chromophores are achiral in many cases, and therefore have no CD, or are separated from the nearest chiral center by several bonds about which relatively free rotation can occur, and therefore have only weak CD. The extrinsic or induced CD observed in the visible and near-UV spectra of the proteins can provide useful information about the conformation and/or environment of the bound chromophore, which usually plays a critical role in the function of the protein. 

Biological systems (e.g., proteins, cells) excited at wavelengths below 500 nm produce considerable autofluorescence that arises mainly from flavins, flavoproteins, NADH, etc. Labels that can be excited at wavelengths above 500 nm are much less susceptible to optical interference from biological chromophores in the test sample. 

Flavin binding proteins. Flavin-binding chromoproteins are collectively referred to as flavoproteins. Their chromophores are derived from riboflavin (vitamin B2 Fig. 15), which is phosphorylated by the flavokinase enzyme to yield flavin mononucleotide (FMN) and, in a second ATP-dependent reaction, FAD pyrophosphorylase attaches an AMP... 

So far, examples to illustrate experimental methods for following the time course of the approach to steady states and of their kinetic interpretation have been restricted to enzymes which do not have a natural chromophore attached to the protein although reference has been made to the classic studies of Chance with peroxidase (see p. 142). Qearly the application of these techniques to the study of enzymes with built in chromophores, such as the prosthetic groups riboflavine, pyridoxal phosphate or haem, contributed considerably to the elucidation of reaction mechanisms. However, the progress in the identification of the number and character of intermediates depended more on the improvements of spectral resolution of stopped-flow equipment than on any kinetic principles additional to those enunciated above. This is illustrated, for instance, by the progress made between the first transient kinetic study of the flavoprotein xanthine oxidase by Gutfreund Sturtevant (1959) and the much more detailed spectral analysis of intermediates by Olson et al. (1974) and Porras, Olson Palmer (1981). 

SERRS has been used successfully to obtain spectra of chromophores in smaller proteins (cytochrome c ) or proteins that contain a higher chromo-phore/polypeptide ratio than GO (hemoglobin ). Among the flavoproteins, flavodoxin, with a MW 16,000 and one flavin mononucleotide (FMN) cofactor positioned near the periphery of the protein, is an ideal protein for SERRS studies. Initial results have shown that higher quality SERRS spectra of this protein may be obtained at low temperatures. No free flavin interference is observed because the protein is apparently stabilized under these conditions. Figure 7 showed the SERRS spectrum of flavodoxin at liquid N2 temperature. It is obvious that the spectrum is quite different from that of FAD or from that reported previously for flavodoxin at room temperature . The spectrum appears to arise from protein-bound flavin based upon a comparison of the observed peak... 

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