Biochemistry - The Chemical Reactions of Living Cells Volume 2 - D. Metzler 1980

Coenzymes - special natural specialized reagents
Flavin coenzymes
Semireduced flavins

One of the most Characteristic Properties of flavins is their ability to accept a single electron, yielding a radical or semiquinone. When the oxidized form of flavin (F) is mixed with its reduced form (FH₂), a single hydrogen atom is transferred from FH₂ to F, resulting in The formation of two FH radicals.

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The equilibrium represented by this equation is independent of pH. However, because all three flavin forms possess distinct pK_a values (Fig. 8-16), the apparent equilibrium constants—which combine the sum of the concentrations of the oxidized, reduced, and radical forms—vary with pH [120, 121]. The proportion of radicals is higher at both acidic and alkaline pH values compared to neutral pH. For 3-alkylflavin [120], the radical formation constant K_f is 2.3∙10^-2, whereas for riboflavin [121] it is 1.5 ∙ 10^-2. Knowing these values alongside the pK_a values shown in Fig. 8-16 makes it possible to estimate the radical concentration at any given pH.

Uncharged flavin radicals are blue in color (with an absorption maximum wavelength λ_max of ~580 nm); however, protonation at N-1 or proton dissociation from N-5 yields red-colored cation or anion radicals with λ_max ≈ 470 nm. (For the complete spectrum, see the book by Mahler and Cordes [122].) Both blue and red radicals are observed in Enzymes, with some enzymes favoring one radical and others favoring the alternative. Hemmerich suggested that enzymes forming red radicals contain a group that forms a strong Hydrogen bond with the proton at position 5. This enhances the basicity of N-1, thereby promoting its protonation and the Formation of the red cation radical.

FIG. 8-16. Properties of oxidized, semireduced, and fully reduced flavins (see Müller et al. [120]).

If an enzyme binds the flavin radical significantly tighter than either the fully oxidized or reduced forms, the reduction of the flavoprotein proceeds in two one-electron steps. In such Proteins, the E^0' values for the two steps may differ substantially. For instance, it has been proposed that a small electron-carrying flavodoxin (azotoflavin) from *Azotobacter* participates in N₂ fixation (Chap. 14, Sec. A,2). Azotoflavin forms a blue semiquinone and exhibits E^0' values of —0.270 V and —0.464 V at pH 7.7, respectively [123]. The latter value represents the lowest redox potential known for Flavoproteins.

Similar flavoproteins have been isolated from anaerobic Bacteria and blue-green Algae (phytoflavin). Determination of the three-dimensional structures of two flavodoxins revealed that the tightly bound riboflavin-5'-phosphate [124, 125] is partially embedded near The surface of a polypeptide composed of 138 Amino Acids. An aromatic amino acid side chain—either Tryptophan or Tyrosine—lies adjacent to the flavin on the protein surface. Surprisingly, in clostridial flavodoxin, the cofactor's phosphate group is not attached to cationic Arginine or Lysine side chains, but rather to a cluster of neutral polar groups comprising four hydroxyl groups from Serine and Threonine residues and four polypeptide chain NH groups [124]. Crystals can be prepared for all three forms of flavodoxins: oxidized, fully reduced, and semiquinone. The flavin semiquinone, much like the oxidized flavin, adopts a virtually planar conformation within the crystal. In all flavodoxins, the two reduction stages are well separated. For example, the flavodoxin from *Peptococcus elsdenii* exhibits E^0' values of —0.115 V and —0.373 V at pH 7.