Flavin, generally reductive reactions

As shown in Figure 2.15, the riboflavin moiety of flavoproteins can undergo two reduction reactions. It can accept one hydrogen, to form the flavin radical (generally written as flavin-H ), followed by a second hydrogen, forming fully reduced flavin-H. ... 

Last, let us consider the possibility of a mechanism other than a hydride transfer in NAD chemistry. Indeed, G. A. Hamilton argued that if a direct hydride transfer process occurs in dehydrogenase reactions, it is unique in biology since proton transfer would be more favorable (279). However, it is not a simple task to distinguish between these two possibilities. Generally, it is simpler to say that the reduction reaction is analogous to a transfer of two electrons rather than postulating a hydride ion. More will be said on this subject in Section 7.1.3 on flavin coenzyme. 

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. 

It is generally agreed that the rate-limiting step is the transfer of reducing equivalents from substrate to the enzyme, that the initial reaction rate is first order with respect to substrate concentration, that flavin is the first electron acceptor (298-300), and that the transfer of electrons from flavin to the heme occurs intramolecularly (300). Anaerobic titration with L-lactate has indicated that the enzyme accepts three electrons (SOI). It has also been shown by EPR studies that upon reduction of the enzyme with L-lactate, a flavin semiquinone is formed to the extent of about 20% of the flavin content of the enzyme (301). However, it is not known whether the flavin semiquinone is a kinetic intermediate during enzyme catalysis. 

Spectral studies of rapid-quench experiments indicate that the substrate/oxidized flavin/O2/reductase combination forms an initial reactive intermediate that subsequently oxygenates the substrate.30,31 These results in combination with the bonding arguments of Chapter 3 prompt the formulation in Scheme 6-7 of a general reaction mechanism for flavin mono-oxygenases. In the absence of substrate the system reduces O2 to HOOH. In either case reduction is by H-atom transfer rather than by electron transfer. 

Since both alcoholic oxidation and O2 reduction are two-electron processes, the catalytic reaction is conceptually equivalent to a transfer of the elements of dihydrogen between the two substrates. Biological hydrogen transfer generally involves specialized organic redox factors (e.g., flavins, nicotinamide, quinones), with well-characterized reaction mechanisms. Galactose oxidase does not contain any of these conventional redox factors and instead utilizes a very different type of active site, a free radical-coupled copper complex, to perform this chemistry. The new type of active site structure implies that the reaction follows a novel biochemical redox mechanisms based on free radicals and the two-electron reactivity of the metalloradical complex. 

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