1.5- Dihydroflavin

The method is quite effective, but is not widely used now because of the ubiquity of digital computers. Zuman and Patel - 36. show circuit designs for some kinetic schemes. Williams and Bruice made good use of the analog computer in their study of the reduction of pyruvate by 1,5-dihydroflavin. In this simulation eight rate constants were evaluated variations in these parameters of 5% yielded discemibly poorer curve fits. 

Flavin coenzymes can exist in any of three different redox states. Fully oxidized flavin is converted to a semiqulnone by a one-electron transfer, as shown in Figure 18.22. At physiological pH, the semiqulnone is a neutral radical, blue in color, with a A ax of 570 nm. The semiqulnone possesses a pAl of about 8.4. When it loses a proton at higher pH values, it becomes a radical anion, displaying a red color with a A ax of 490 nm. The semiqulnone radical is particularly stable, owing to extensive delocalization of the unpaired electron across the 77-electron system of the isoalloxazine. A second one-electron transfer converts the semiqulnone to the completely reduced dihydroflavin as shown in Figure 18.22. 

Eckstein, J. W., and Ghisla, S. (1991). On the mechanism of bacterial luciferase. 4a,5-Dihydroflavins as model compounds for reaction intermediates. In Flavins Flavoproteins, Proc. Int. Symp., 10th, 1990, 269-272. 

Dioxygenases often have broad substrate specificity and require only a minimal characteristic structure for substrate recognition [310], Transition metal or an organic cofactor mediates dioxygen activation needed by the oxygenases action. Iron and copper, in their lower oxidation states are the metals most commonly used, but also organic co-factors like dihydroflavin and tetrahydropterin are able to activate the oxygen molecule. 

Figure 5.3 The flavin coenzymes FAD and FMN. Note that in contrast to NAD+, flavins can be half-reduced to the stable radical FADH or fully reduced to the dihydroflavin shown.

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... 

Table 5. Comparison of the Molar Extinction Coefficients at 450 nm of 1,5-Dihydroflavins in the...

Flavohydroquinone bound to apoproteins plays a very important role in flavo-protein-catalysis, either in the electron-transfer to substrates or other enzymes or in the oxygen activation reaction. The chemical reactivity of 1,5-dihydroflavin bound to apoproteins can differ drastically from that of free flavin. The reactivity is likely governed by factors such as the conformation of the bound flavohydroquinone and the ionization state (cf. below). 

Dihydroflavin exhibits rather diffuse visible absorption characteristics (cf. Table 4). In contrast, the derivatives (20), (21), (23) and (24) show well-defined spectra. The absorption maxima occur at 430 nm ((20)), 360 nm ((21)), 400 nm... 

Examples of this class of enzymes are glucose oxidase and D-amino acid oxidase The classification of the flavoproteins used here is that originally suggested which has been modified recently . In the author s own view the original classification has the advantage of being simple and yet quite useful whereas the new classification does not add to simplify and classify the rather complex picture of flavoprotein catalysis. Nevertheless, in flavoprotein oxidases, the 1,5-dihydroflavin is very reactive towards Oj. On the other hand, the two-electron reduced form of flavoprotein oxidases reacts slowly with pure one-electron acceptors, e.g. ferricyanide. That the two-electron transition is biologically favoured in these enzymes explains why they can react easily with sulfite... 

Molecular oxygen usually reacts rapidly with only those organic substrates, such as dihydroflavins, that are able to form stable free radicals. However, the endiolate anion of Eq. 13-50 may be able to donate a single electron to 02 to form a superoxide-organic radical pair prior to formation of the peroxide (see also Eq. 15-30). Similar oxygenase side reactions have been observed for a variety of other enzymes that utilize carbanion mechanisms.283 The reaction of rubisco with 02 is of both theoretical and practical interest, the latter because of its significance in lowering the yield in photosynthesis (Chapter 23). 

Unusual features of riboflavin as recorded by some researchers include (1) High levels in liver inhibit tumor formation by azo compounds in animals (2) free radicals are formed by light or dehydrogenation flavine semiquinone dihydroflavin+ (3) free vitamin is found only in retina, urine, milk, and semen (4) substitution of adenine by other purines and pyrimidines destroys activity of flavin adenine dinucleotide (FAD) ... 

Hence, bases (HO-, Oj, e q) catalyze the autoxidation of hydrazines, di-hydrophenazine (H2Phen), and dihydroflavins (H2F1) ... 

This is a convenient system for the synthesis of HOOH and may be similar to the mechanistic path for HOOH synthesis in biology via dihydroflavin proteins. Aniline (PhNH2) and phenylhydrazine (PhNHNH2) in combination with HO-and 02 are effective reagents for the in situ synthesis of superoxide ion 16... 

These H-atom transfer cycles closely resemble those of 3-methyl-lumiflavin and riboflavin, and may represent the redox mechanisms of flavin/hydroflavin/ dihydroflavin proteins in biology. 

When searching for intermediates in a chemical reaction it is useful to examine the reaction in both the forward and reverse directions. The reduction of pyruvic acid by dihydroflavin is the retrograde of the... 

Reactions were studied under the pseudo first-order condition of [substrate] much greater than [initial dihydroflavin]. Under these conditions, the reactions are characterized by a burst in the production of Flox followed by a much slower rate of Flox formation until completion of reaction. The initial burst is provided by the competition between parallel pseudo first-order Reactions a and b of Scheme 3. These convert dihydroflavin and carbonyl compound to an equilibrium mixture of carbinolamine and imine (Reaction a), and to Flox and alcohol (Reaction b), respectively. The slower production of Flox, following the initial burst, occurs by the conversion of carbinolamine back to reduced flavin and substrate and, more importantly, by the disproportionation of product Flox with carbinolamine (Reaction c followed by d). Reactions c and d constitute an autocatalysis by oxidized flavin of the conversion of carbinolamine back to starting dihydroflavin and substrate. In the course of these studies, the contribution of acid-base catalysis to the reactions of Scheme 3 were determined. The significant feature to be pointed out here is that carbinolamine does not undergo an elimination reaction to yield Flox and lactic acid (Equation 25). The carbinolamine (N(5)-covalent adduct) is formed in a... 

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