Oxidized flavin, free

He/minthosporium (15). The mode of action is considered to be inhibition of the enzyme NADPH-cytochrome C reductase, which results in the generation of free radicals and/or peroxide derivatives of flavin which oxidize adjacent unsaturated fatty acids to dismpt membrane integrity (16) (see Enzyme inhibitors). 

XO, which can excite stable triplet oxygen (302), is a pro-oxidant. Milk which undergoes spontaneous rancidity contains about 10 times the normal level of XO, and spontaneous oxidation can be induced in normal milk by the addition of XO to about four times normal levels. Heat-denatured or flavin-free enzyme is ineffective and the susceptibility of unsaturated fatty acids to oxidation increases with the degree of unsaturation. 

In sharp contrast to the reductive half-reaction, where the free oxidized flavin is totally inert in the presence of physiological substrates, reduced model flavins are appreciably reactive (nonenzymatically) with O2 and other electron acceptors. However, the O2 reactivity of reduced flavin is complicated for two perhaps related reasons (61). First, the reaction is autocatalytic owing to the formation of 2F (from F and FH2) which in its anionic state is extremely reactive with Oo. Second, the superoxide radical is an important kinetic intermediate in O2 reduction (59). Neither of these features is observed with the reduced flavoprotein oxidases. 

However, these experiments may not have established a mechanism for natural flavoprotein catalysis because the properhes of 5-deazaflavins resemble fhose of NAD+ more fhan of flavins. Their oxidation-reduction pofentials are low, they do not form sfable free radicals, and fheir reduced forms don f reacf readily with O2. Nevertheless, for an acyl-CoA dehydrogenase the rate of reaction of the deazaflavin is almost as fast as that of nafural EAD. Eor fhese enzymes a hydride ion fransfer from fhe P CH (reaction type D of Table 15-1) is made easy by removal of the a-H of the acyl-CoA to form an enolafe anion intermediate. 

The separation of intermediate II from the flavin obtained is shown in Fig. 43. The free oxidized flavin (450 nm) is well separated from the protein (280 nm). The latter presumably includes both intermediate II (370 nm) and any unreacted enzyme or other contaminating proteins. The oxidized flavin presumably includes both that which formed the nonenzymic pathway and any that had already been formed as product of the enzymic pathway before the reaction was stopped. 

Oxygenases can be classified into three groups based on the co-factor required for catalytic activity of the enzyme. One is the transition-metal free enzyme containing an organic prosthetic group such as flavin, but oxidation of unactivated C-H bonds by this enzyme has not yet been found. The other two are metalloenzymes containing copper or iron. Despite the fact that these enzymes are largely distributed in nature, the molecular mechanisms of their reactions are known in considerably less detail. Elucidation of this... 

Chorismate Synthase. - Chorismate synthase catalyses the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh and last enzyme of the shikimate pathway. Chorismate constitutes a major building block for the biosynthesis of an array of aromatic compounds, including the amino acids phenylalanine, tryptophan and tyrosine. Although this reaction does not involve a change in redox states, the enzyme requires reduced FMN for activity, and binds oxidized flavin only very weakly which results in its isolation as the flavin-free apo-enzyme. Macheroux and co-workers have used spectrophotometry, fluorimetry and EPR and ENDOR to investigate binding of the oxidized, reduced and radical forms of FMN to chorismate synthase in the presence of (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate(a substrate ana-... 

At the initial step electrons are transferred from NADPH to the oxidized FAD, reducing it to FADH2. Disproportionation between flavins leads to the formation of two free radicals FADH and FMNH. Electron transfer from FMNH to the heme results in the reduction of Fe3+ to Fe2+, and the reduced heme becomes able to bind 02 to form the intermediate... 

FIGURE 32-7 Sources of free radical formation which may contribute to injury during ischemia-reperfusion. Nitric oxide synthase, the mitochondrial electron-transport chain and metabolism of arachidonic acid are among the likely contributors. CaM, calcium/calmodulin FAD, flavin adenine dinucleotide FMN, flavin mononucleotide HtT, tetrahydrobiopterin HETES, hydroxyeicosatetraenoic acids L, lipid alkoxyl radical LOO, lipid peroxyl radical NO, nitric oxide 0 "2, superoxide radical. 

In the reduction or oxidation of quinone/ quinol systems, free radicals also appear as intermediate steps, but these are less reactive than flavin radicals. Vitamin E, another qui-none-type redox system (see p.l04), even functions as a radical scavenger, by delocalizing unpaired electrons so effectively that they can no longer react with other molecules. 

The absorption spectral properties of the neutral and anionic forms are quite different as shown in Fig. 1. Due to the rapid dismutation of flavin radicals to form an equilibrium mixture with the hydroquinone and oxidized forms of the flavin, special procedures must be employed to measure the spectral properties of free flavin radicals. Nearly quantitative amounts of anion radical can be formed in aprotic solvents under basic conditions Alkylation of the N(5) position of the flavin hydroquinone followed by oxidation results in nearly quantitative formation of the... 

In contrast to their instability free in solution, flavin semiquinones bound to proteins are generally (although there are exceptions) quite stable and can be generated in nearly quantitative yields. This stability results from thermodynamic considerations in which the oxidized/semiquinone oxidation-reduction couple is usually more positive and well separated from the semiquinone/hydroquinone couple. In addition to thermodynamic stabilization, kinetic stabilization of protein-bound semiquinones has also been observed and will be discussed in more detail in subsequent portions of this article. 

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