Flavoprotein absorption bands

Like the nicotinamide coenzymes (Fig. 13-15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to assay reactions involving a flavoprotein. 

The name cytochrome bs is now generally used for a protohemoprotein which exists in high amounts in the endoplasmic reticulum of mammalian liver cells. It shows an asymmetrical a-absorption band with a peak at 556 nm and a shoulder around 560 nm in the reduced state. However, there has been some confusion about its nomenclature. This cytochrome is firmly bound to the membrane structure and reduced with NADH by a flavoprotein (cytochrome bs reductase) which is also bound to the membrane. It is solubilized by proteolytic cleavage from the membrane and has been crystallized from several sources. The molecular weight of such preparations are about 11,000. Although both primary and ternary structures of such preparations are known, its biological role is still uncertain. Earlier findings have been fully reviewed by Strittmatter (94)-... 

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. 

Since no further increase of activity could be achieved, the authors consider the enzyme as essentially pure. The final preparation shows no absorption bands (125) or fluorescence (39). (Cohen, 61, found trans-aminating activity to be associated with protdn fractions exhibiting green fluorescence of the flavoprotein type.) LenArd and Straub s preparation, in contrast to those of Kritzmann and Cohen, fails to effect the reaction GLp AS. This important fact indicates either the presence in the latter preparations of two distinct aminopherases, aphcL AL and aphoL AS,... 

A much more active L-amino acid oxidase, discovered in snake venom by Zeller, has been isolated by Singer and Kearney and identified as a flavoprotein with FAD as the prosthetic group. The enzyme has a molecular weight of about 62,000, and the spectrum shows absorption bands at 389 and 465 m/x. With L-leucine as the substrate, the turnover number at 38° is 3100 moles of substrate oxidized per minute per mole of flavin even crude venom has higher activity than the purified rat liver enzyme. Snake venom L-amino acid oxidase exists in active and inactive forms which are interconvertible under the influence of pH and inorganic ions. 

It had been suggested by Franke and Deffner that this enzyme was a flavoprotein. Notatin has absorption bands at 377 and 455 m, and the prosthetic group was identified as FAD. However, the process of splitting the enzyme appears to denature the protein, and it has not been possible to establish that FAD will restore the catalytic activity. The molecular weight has been determined by sedimentation measurements to be 152,000,... 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

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