1
Structure and Properties of Flavins

Tetiana Pavlovska and Radek Cibulka

University of Chemistry and Technology, Prague, Department of Organic Chemistry, Technická 5, Prague, 166 28, Czech Republic

1.1 Introduction and History of Flavins Discovery

Along with the historical events in 1937 and 1938, such as the explosion of the Hindenburg, the unsolved disappearance of Amelia Earhart, and the inexorable forthcoming of another world war, the world of chemistry was marked by two Nobel Prizes in the discovery and characterization of vitamins. Paul Karrer in Zurich and Richard Kuhn in Heidelberg succeeded almost concurrently in determining the structure of vitamin B2 (riboflavin), which made it possible to produce the vitamin by artificial means [1]. Both scientists received the Nobel Prize for their research on vitamins, Paul Karrer in 1937 for his investigations on carotenoids, flavins, and vitamins A and B2 [2], and Richard Kuhn in 1938 for his work on carotenoids and vitamins [3].

Long before the first synthesis of riboflavin, it had been described as a component of cow milk by an English chemist, A. Wynter Blyth, in 1879. He isolated a bright yellow pigment, which he called lactochrome [4]. It was later described in several reports as a water-soluble substance with green fluorescence found in milk, malt, eggs, liver, and pig heart [5]. Subsequently, in the late 1920s and early 1930s, a significant amount of research in this area was undertaken. The most intensive research outbreak started when it was found that the yellow pigment was a constituent of the vitamin B complex, which plays a vital role in living creatures [6]. The name riboflavin was given to replace the variety of names previously used (lactoflavin, ovoflavin, hepatoflavin), which were related to the source from which the pigment was isolated. Riboflavin represents the D-ribityl derivative of the isoalloxazine heterocycle, whose yellow color gives the second part of the name (from Latin: Flavus = yellow).

Several groups contributed to identification of the first flavin cofactor (flavin mononucleotide [FMN] or riboflavin-5´-phosphate) [5, 7]. Kuhn and co-workers synthesized FMN [7a] and mentioned that it is identical with the "cytoflav" discovered by Banga and Szent-Györgyi [7b]. Almost at the same time, Warburg and Christian isolated a yellow protein from yeast. Theorell found that "yellow enzyme" consisted of two parts: flavin plus a phosphate group and aprotein called apoenzyme [7c,d]. The second identified flavin cofactor, flavin adenine dinucleotide (FAD), was isolated as a cofactor of D-amino acid oxidase by the same research group in 1938 [8]. In 1954, the structure of FAD was proven by total synthesis [9]. These milestones established riboflavin as a vitamin B2, and FMN and FAD as cofactors in enzymatic catalysis [10]. The phenomenon of covalent attachment of the flavins to proteins was first established in mammalian succinate dehydrogenase in 1955 [11]. Since then, the covalent attachment of the flavins to proteins has been demonstrated through the C6 atom and C8 methyl group (see Figure 1.1 for numbering) of the flavin in many flavoproteins (see Chapter 2). In 2005, X-ray crystallography was used to reveal the first example of a flavin bicovalently linked to a protein in glucooligosaccharide oxidase [12].

Figure 1.1 The structure of benzo[g]pteridine (1), alloxazine (2), isoalloxazine (flavin, 3), and their biologically relevant derivatives.

Flavins and flavoproteins have been found to play a crucial role in a myriad of metabolic pathways (see Chapter 2 for details). They are involved in aerobic metabolism by catalyzing the two-electron dehydrogenation of various substrates and are responsible for one-electron transfer to different metal centers via their radical states [13]. Flavoproteins play a significant role in soil detoxification processes via the hydroxylation of aromatic compounds, forming parts of multi-redox-center enzymes, such as nicotinamide adenine dinucleotide (NADH) dehydrogenase, xanthine oxidase/dehydrogenase, and cytochrome P450. Flavins take part in the production of light in bioluminescent bacteria and are involved in blue light-initiated reactions, such as plant phototropism and nucleic acid-repair processes, the regulation of biological clocks, energy production, biodegradation, chromatin remodeling, apoptosis, and protein folding [14]. Flavin-dependent light-responsive proteins and enzymes, notably DNA photolyases, cryptochromes, and light-oxygen-voltage (LOV) and blue-light sensors using flavin adenine dinucleotide (BLUF) domains participate in many critical biological processes, including DNA repair, the photoregulation of circadian rhythms, and gene expression [15].

In this chapter, the compounds from the "flavin family" and their properties are reviewed, with a special focus on those, which may be of importance from the viewpoint of flavin catalysis: redox and acid-base properties, reactivity with nucleophiles and electrophiles, and noncovalent interactions. Chapter 2 describes the behavior of flavin derivatives in natural systems. The spectral properties of flavin derivatives are reviewed in detail in Chapter 3. There are also several excellent books focusing on various aspects of the flavins and flavoenzymes [16-18].

1.2 The Structure of the Flavins and Their Derivatives

According to Massey, the flavins are a class of yellow, water-soluble chemical compounds containing a heterocyclic 7,8-dimethylisoalloxazine ring, which include riboflavin, FMN, and FAD [10]. However, the flavin family has now been extended to all compounds containing an isoalloxazine nucleus as well as the alloxazines, 5-deazaflavins, and other derivatives such as their corresponding N-oxides and flavinium salts.

The underlying heterocyclic structure of flavin molecules is benzo[g]pteridine (1; Figure 1.1). Its 2,4-dioxo derivatives are alloxazines with the most cited compound being lumichrome (2a). However, the biologically most important molecules, such as lumiflavin (3a), riboflavin (3b), FMN (3c), and FAD (3d), are derived from the tautomeric isoalloxazine. Iso- and alloxazines are analogous compounds; however, their spectroscopic and photophysical properties are different (see Chapter 3). Notably, isoalloxazines exhibit intense fluorescence and relatively long fluorescence lifetimes with fluorescence quantum yields one order of magnitude higher than those observed for the alloxazines [19].

Early interest in the photophysical and photochemical properties of the alloxazines 2 was mainly driven by bearing resemblance to the flavins 3. It is also important to assess the toxicity of lumichromes as products formed during the photochemical reactions of riboflavin. Alloxazines, as products of the biochemical, chemical, or photochemical decomposition of biologically active isoalloxazines, are present in the majority of biological tissues [20]. Lumichrome (2a), for example, has been found to inhibit flavin reductase in living Escherichia coli cells [21].

The flavins take part in both two-electron processes, such as the oxidation of organic compounds in prokaryotic and eukaryotic respiratory chains, and one-electron transfer in cytochromes and other redox centers [22]. Another key property of the flavin cofactors is their ability to form adducts with different substrates [23]. However, flavin adducts similar to those formed in nature are hard to synthesize, which can be attributed to their low stability outside of an enzyme. The stability of flavin adducts can be increased by introducing substituents at the N5 position [24]. Thus, flavinium salts have been established to mimic the functions of flavin-dependent monooxygenases. Flavinium salts 4·X possess unique biomimetic organocatalytic properties, which promote various chemoselective oxidative reactions under mild conditions and can be derived both from isoalloxazines 4a·X and alloxazines (4b·X and 4c·X) [25]. A rare example of a 1,5-diblocked quinoid flavinium salt 4d·X2 was expected to possess a high free-energy content and very positive redox potential, which is, however, at the expense of its stability (Figure 1.2) [26].

Figure 1.2 The general structures of isoalloxazinium and alloxazinium salts.

Source: Modified from Eberlein and Bruice [26].

In addition to the flavins (isoalloxazines), structurally similar 5-deazaflavines with the N5 atom replaced by a methine group also occur in biological systems (Figure 1.3) [27]. The 5-deazaflavin family includes 8-hydroxy-7-desmethyl-5-deazariboflavin coenzymes F420 (5b) and F0 (5a). F420 was isolated by Wolfe and coworkers in 1978 from various methanogenic bacteria, in which it is involved in the key steps of methanogenesis and carbon assimilation, and represents a derivative of F0 bearing a lactyl-oligoglutamyl group attached via a phosphodiester linkage to the ribityl side chain (Figure 1.3) [28]....