Flavins physical properties

The following is review on the molecular and physical properties of this class of monooxygenases, which are also known as hydroxylases. A typical monooxygenase reaction is the hydroxylation of an alkane to an alcohol which involves a reduced cosubstrate that reduces a second atom within the O2 molecule to form water. Flavin-containing monooxygenases include lysine oxygenase and 4-hydroxybenzoate hydroxylase. Reduced pteri-dines are involved in the phenylalanine hydroxylase and tryptophan hydroxylase reactions. See also Cytochrome P-450... 

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... 

Today, four kinds of 8a-substituted peptides are known (Scheme 2). The published data up to 1976 were summarized recently . The chemical synthesis of the various flavin peptides and their physical properties are described in the references given in Table 2 and were reviewed recently . ... 

The 8a-modified flavins show some remarkable physical properties which differ from those of common flavins. Since these properties are very helpful in the identification of modified 8a-substituted flavocoenzymes originating from biological materials the physical properties are presented briefly here. Compared to riboflavin (Scheme 2, (7), R = H) the visible light absorption properties of (2) to... 

The most prominent feature of the chemistry of flavin is its redox properties. These properties make flavin especially suitable for its broad involvement in biological reactions. In the following the pH-dependent species formed in one- and two-electron reductions will be dealt with first, including their visible absorption and fluorescence properties. These physical properties form the basis of many kinetical and analytical studies. In Scheme 3 the structures refer to the free and protein-bound prosthetic groups (cf. Scheme 1). To study the physical properties of the flavocoenzymes often N(3)-alkylated lumiflavin (R = CH3) is used which is better soluble in a variety of solvents. Other physical and chemical properties of these species will be discussed subsequently. 

Reduction of flavin by two electrons yields the 1,5-dihydroflavin (Scheme 3), often called reduced flavin . Since isomeric two-electron reduced flavin structures are known (cf. below), the term reduced flavin should be avoided unless defined to prevent misunderstanding. From all flavin species possible in a redox reaction the solution of 1,5-dihydroflavin is, in contrast to that of some isomeric compounds, devoid of a stront colour but not colourless as indicated by the term leucoflavin , which is still used (Table 4). The only true colourless species is (77). Because of the very high oxygen-sensitivity of 1,5-dihydroflavin its chemical and physical properties were investigated only recently Long before crystallographic data on flavins were available, conclusions were drawn from the molar extinction coefficient at 450 nm of 1,5-dihydroflavins with respect to the planarity of the molecules. From the data presented in Table 5 it was proposed that anionic... 

The ongoing research into the structure and mechanism of flavoenzymes has been the subject of several recent excellent reviews The proceedings of six symposia held at intervals over the past 16 years provide an overall perspective on the progress of flavoenzyme research over this time period. The intent of this article will be to focus directly on the chemical and physical properties of the semiquinone form of flavin coenzymes to the extent that current knowledge permits, from the point of view of both model system studies and from existing knowledge of their properties in flavoenzyme systems. For an in-depth treatment of flavin and flavoenzyme redox properties in which the oxidized and hydroquinone forme as well as the semiquinone form are discussed as related to their biological function, the reader is refered to the article by F. Muller in this volume. 

This Report follows the pattern established by the previous Reporter in Volume 1. The section dealing with fused 5,6-systems is chiefly concerned with the chemistry of purine and purine analogues and that on fused 6,6-systems with the chemistry of pteridines and flavins. Within the discussion of a given ring system, reference is made to synthetic methods before physical properties and reactivity. Where appropriate, fully aromatic compounds are discussed before those of an increased level of saturation. Because of the limits of space imposed on the Senior Reporters of this volume, it has been necessary to omit reference to much interesting work. 

Metallo-Flavoproteins. As was mentioned in the case of cytochrome reductase, enzymes are known that contain metal cofactors in addition to flavin. These are called metallo-flavoproteins. The presence of metals introduces complexity into the reaction, since the metals involved, iron, molybdenum, copper, and manganese, all exist in at least two valence states and can participate in oxidation-reduction reactions. The enzymes known to be metallo-flavoproteins include xanthine oxidase, aldehyde oxidase, nitrate reductase, succinic dehydrogenase, fatty acyl CoA dehydrogenases, hydrogenase, and cytochrome reductases. Before these are discussed in detail some physical properties of flavin will be presented. 

Physical Properties of Flavins. The most striking characteristic of riboflavin and its derivatives is the yellow color. This absorption spectrum reveals three major peaks, at 260 m/i, 375 mju, and 450 m. Both the 375 m and 450 m/u peaks disappear on reduction. This change in absorption permits the extent of oxidation or reduction to be measured spectrophotometrically or visually on reduction the yellow color disappears as the leuco form is produced. The reaction is thought to be as indicated in (II). Eo (pH 0) for simple flavin derivatives is —0.187 volts,... 

As is borne out by these data, uncertainty about the site of substrate or inhibitor addition to the flavin in photochemistry and in enzymology is very similar. Moreover the adduct chromophores are absolutely identical in the photochemical and in the enzymic cases (159, 119). We may be far away from explaining these phenomena as functions of the molecular structures and physical properties of flavin, substrate and environment, but the model relevance of flavin photochemistry to a molecular understanding of flavin enzymology cannot be denied. 

The structure of the modified flavocoenzymes was elucidated by chemical synthesis and comparative physical studies 126, iso) (Scheme 2, (7), (S)). The compounds possess some unusual properties some of which are collected in Table 3. The most prominent difference between (7) and (S) is the fluorescence behaviour (7) is fluorescent, (5) does not fluoresce. Moreover, at pH > 10 the fluorescence quantum yield of (7) increases by a factor of about 2, in contrast to normal flavin the fluorescence of which is quenched. By this property (7) and (5) are easily distinguished (Table 3). From the visible absorption properties of analogs of (7) and (5) it was concluded that both compounds can exist in two tautomeric forms (proton on N(l) or C(8)O, C(6)O), leading to quinoid structures. 

Another Japanese research group isolated two riboflavin derivatives from the culture filtrate of Schizophyllum commune and called these natural products Schizo-flavin 1 and 2 The physical and chemical properties of these compounds are... 

Cheeseman et al. reported in 1972 the purification and properties of a fluorescent compound from Methanobacterium. Owing to the first visible absorption at 420 nm the unknown compound was called Factor-420. It was not until 1978 till it became clear that the compound is a flavin-like molecule By different physical techniques it could be shown that the molecule is a 5-deaza-FMN derivative (Scheme 2, (70)) where at position 8 the methyl group is replaced by a hydroxy group. In addition, the side chain phosphate group is esterified by a lactyl group which, in turn, is linked to a diglutamyl moiety via a peptide bond. Factor-420 functions as... 

It should be obvious from the literature discussed in this article that progress in our understanding of the properties of flavin semiquinones and their role in flavoenzyme catalysis has increased dramatically over the past twenty years. This has been due to the application of sophisticated chemical and physical approaches, as well as to an increase in the number and diversity of flavoenzymes which have been purified to homogeneity in quantities sufficient for extensive study. 

Table 8. Physical-chemical properties of NbF and TaF5 (after Galkin [71] and Rabinovich and Flavin [72]).

Carotenoids vs. Flavins. To date, considerable evidence supports the contention that carotenoids are involved in the light-activating mechanism of DPE s O, 4, 5, 7-10). From a purely physical view, a flavin can be perceived as a more likely candidate for activation of DPE s than can a carotenoid. The following molecular properties that favor riboflavin over... 

Two forms of nitrite reductase have been isolated from scutella, roots and etiolated leaves of maize (Hucklesby et al., 1972 Dalling et al., 1973). The physical and biochemical characteristics of one form are nearly identical with those of the enzyme from the green leaf. Only one form of the enzyme was found in the green leaf. Except for differences in thermal stability, and ion charge, the properties of the second form are nearly identical with that of the enzyme from green leaves. The enzymes from the nonchlorophyllous tissue, like the enzyme from green leaves, can utilize reduced dyes or ferredoxin but not nicotanamide or flavin nucleotides as electron donors. 

The phosphorylated and non-phosphorylated forms of vitamin Bg have various physical and chemical properties. Vitamin Bs in the form of pyridoxal-5 -phosphate (PLP) and to a lesser extent, pyridoxamine-5 -phosphate (PMP), functions as a coenzyme in over 100 enzymatic reactions. All the forms of vitamin Be possess vitamin activity because they can be converted in vivo to pyridoxal. PN, PM and PL are converted to 5 -phosphate by a single kinase enzyme which in the brain and liver is most active with zinc. PNP and PMP are then converted to PLP by flavin dependent oxidase this is the reason why vitamin B2 deficiency causes a fall in available PLP (Holman 1995). Human cells can synthesize PLP from three vitamers via the Bg salvage pathway but cannot synthesize PLP de novo and must obtain it from dietary sources. 

Flavins are versatile protein cofactors when inserted into proteins, as these alford a broad range of redox reactions and catalytic properties due to their unique physical and chemical properties. 

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