Flavoprotein radicals

The development of a wide range of special forms of EPR was initiated when the idea of double resonance (using simultaneous irradiation by two different sources) was cast in 1956 by G. Feher at Bell Telephone Labs in his seminal paper on ENDOR, electron nuclear double resonance (Feher 1956). BioEPR applications of ENDOR were later developed on flavoprotein radicals in a collaboration of A. Ehrenberg and L. E. G. Eriksson in Stockholm, Sweden, and J. S. Hyde at Varian in Palo Alto, California (Ehrenberg et al. 1968), and on metalloproteins in a joint effort of the groups of R. H. Sands in Ann Arbor, I. C. Gunsalus in Urbana, Illinois, and H. Beinert in Madison (Fritz et al. 1971). 

Palmer, G., Muller, F., Massey, V. Electron paramagnetic resonance studies on flavoprotein radicals. In Flavins and flavoproteins (Kamin, H. ed.) pp. 123-137. Baltimore, Univ. Park Press 1971... 

Flavoprotein radicals have been extensively studied by ESR [139] and ENDOR [140] spectroscopies. Their ESR spectra, with few exceptions, are broad and featureless because of the slow rate of tumbling of the radicals, even in fluid solution. Nevertheless, some structural information is available based on the width of the observed ESR line. The spectra observed fall into three classes, having widths of about 19,15 and 12 G. Non-covalently bound semiquinones have linewidths of either 19 or... 

Similarly, the reduction of riboflavin prosthetic groups (FP) of some flavoprotein dehydrogenases may also involve a free radical intermediate. 

The splitting of H2 by hydrogenases is heterolytic (into H and H" ), rather than homolytic (into two H. radicals). The hydride is considered to deliver two electrons at a time to the enzyme. A [4Fe-4S] cluster in proteins can, however, accept only one electron. Other redox enzymes, e.g. flavoproteins, dealing with two-electron donors (like NADH) solve this problem by first accommodating both electrons onto the flavin, whereafter these electrons are transferred to an Fe-S cluster one at a time. 

A flavoprotein oxidase, which is also a methanol oxidizing enzyme, was inhibited by cyclopropanol 4 through the formation of a N-5 flavin adduct with a ring opened cyclopropyloxy radical [10]. 

Anderson, R. F. Flavin-oxygen complex formed on the reaction of superoxide ions with flavo-semiquinone radicals. In Flavins and flavoproteins (Massey, V., Williams, C. H. eds.) pp. 278-283, New York, Elsevier North Holland 1982... 

Tertiary amine oxides and hydroxy la mines are also reduced by cytochromes P-450. Hydroxylamines, as well as being reduced by cytochromes P-450, are also reduced by a flavoprotein, which is part of a system, which requires NADH and includes NADH cytochrome b5 reductase and cytochrome b5. Quinones, such as the anticancer drug adriamycin (doxorubicin) and menadione, can undergo one-electron reduction catalyzed by NADPH cytochrome P-450 reductase. The semiquinone product may be oxidized back to the quinone with the concomitant production of superoxide anion radical, giving rise to redox cycling and potential cytotoxicity. This underlies the cardiac toxicity of adriamycin (see chap. 6). 

Due to the variety of enzymatic environments in which FAD/ FADH /FADHZ can be held, enzymes that contain them exhibit redox potentials ranging from -0.45 to + 0.15 V at pH 7 (Bugg, 1997). Xenobiotic compounds whose structures suggest they can be reduced via free radical mechanisms or via hydride transfers may be reduced by such flavoproteins. 

Like the nicotinamide coenzymes (Fig. 13-15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to assay reactions involving a flavoprotein. 

In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble ben-zoquinone with a long isoprenoid side chain (Fig. 19-2). The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains. Ubiquinone can accept one electron to become the semi-quinone radical ( QH) or two electrons to form ubiquinol (QH2) (Fig. 19-2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor. Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement. 

However, these experiments may not have established a mechanism for natural flavoprotein catalysis because the properties of 5-deazaflavins resemble those of NAD+ more than of flavins.239 Their oxidation-reduction potentials are low, they do not form stable free radicals, and their reduced forms don t react readily with 02. Nevertheless, for an acyl-CoA dehydrogenase the rate of reaction of the deazaflavin is almost as fast as that of natural FAD.238 For these enzymes a hydride ion transfer from the (3 CH (reaction type D of Table 15-1) is made easy by removal of the a-H of the acyl-CoA to form an enolate anion intermediate. 

If an enzyme binds a flavin radical much more tightly than the fully oxidized or reduced forms, reduction of the flavoprotein will take place in two one-electron steps. In such proteins the values of E° for the two steps may be widely separated. The best known examples are the small, low-potential electron-carrying proteins known as flavodoxins.266 269a These proteins, which carry electrons between pairs of other redox proteins, have a variety of functions in anaerobic and photosynthetic bacteria, cyanobacteria, and green algae. Their functions are similar to those of the ferredoxins, iron-sulfur proteins that are considered in Chapter 16. 

The enzyme is organized into two structural domains,201 one of which binds FAD and the other NADP+. Similar single-electron transfers through flavoproteins also occur in many other enzymes. Chorismate mutase, an important enzyme in biosynthesis of aromatic rings (Chapter 25), contains bound FMN. Its function is unclear but involves formation of a neutral flavin radical.276 277... 

The presence of metal ions in many flavoproteins suggested a direct association of metal ions and flavins. Although oxidized flavins do not readily bind most metal ions, they form red complexes with Ag+ and Cu+ with a loss of a proton from N-3.278 Flavin semiquinone radicals also form strong red complexes with many metals.264 If the complexed metal ion can exist in more than one oxidation state, electron transfer between the flavin and a substrate could take place through the metal atom. However, chelation by flavins in nature has not been observed. Metalloflavoproteins probably function by having the metal centers close enough to the... 

Pyruvate oxidase. The soluble flavoprotein pyruvate oxidase, which was discussed briefly in Chapter 14 (Fig. 14-2, Eq. 14-22), acts together with a membrane-bound electron transport system to convert pyruvate to acetyl phosphate and C02.319 Thiamin diphosphate is needed by this enzyme but lipoic acid is not. The flavin probably dehydrogenates the thiamin-bound intermediate to 2-acetylthiamin as shown in Eq. 15-34. The electron acceptor is the bound FAD and the reaction may occur in two steps as shown with a thiamin diphosphate radical intermediate.3193 Reaction with inorganic phosphate generates the energy storage metabolite acetyl phosphate. 

Metalloenzymes of at least three different types catalyze the destruction of superoxide radicals that arise from reactions of oxygen with heme proteins, reduced flavoproteins, and other metalloenzymes. These superoxide dismutases (SODs) convert superoxide anion radicals 02 into H202 and 02 (Eq. 16-26). The H202 can then be destroyed by catalase (Eq. 16-8). 

Flavin adenine dinucleotide. See FAD Flavin adenine diphosphate. See FAD Flavin coenzymes 766,780 - 795 modified 788, 789 reduced 794 Flavin radicals 792 color of 794 formation constant 794 Flavocytochrome b2 782, 794, 847 Flavodoxins 793, 799, 800 Flavoprotein(s) 513, 788... 

Two ascorbate radicals can react with each other in a disproportionation reaction to give ascorbate plus dehydroascorbate. However, most cells can reduce the radicals more directly. In many plants this is accomplished by NADH + H+ using a flavoprotein monodehydroascorbate reductase.0 Animal cells may also utilize NADH or may reduce dehydroascorbate with reduced glutathione.CC/ff Plant cells also contain a very active blue copper ascorbate oxidase (Chapter 16, Section D,5), which catalyzes the opposite reaction, formation of dehydroascorbate. A heme ascorbate oxidase has been purified from a fungus. 11 1 Action of these enzymes initiates an oxidative degradation of ascorbate, perhaps through the pathway of Fig. 20-2. 

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