Flavoprotein dehydrogenases mechanisms

The chemistry of flavins is complex, a fact that is reflected in the uncertainity that has accompanied efforts to understand mechanisms. For flavoproteins at least four mechanistic possibilities must be considered.1533 233 (a) A reasonable hydride-transfer mechanism can be written for flavoprotein dehydrogenases (Eq. 15-23). The hydride ion is donated at N-5 and a proton is accepted at N-l. The oxidation of alcohols, amines, ketones, and reduced pyridine nucleotides can all be visualized in this way. Support for such a mechanism came from study of the nonenzymatic oxidation of NADH by flavins, a reaction that occurs at moderate speed in water at room temperature. A variety of flavins and dihydropyridine derivatives have been studied, and the electronic effects observed for the reaction are compatible with the hydride ion mecha-nism.234 236... 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

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. 

The foeus of this chapter is the soluble electron transfer complex formed between the nieotinamide-independent trimethylamine dehydrogenase (TMADH) and eleetron transferring flavoprotein (ETF). Recent studies of this physiological electron transfer complex have provided invaluable insight into (i) the mechanisms of inter and intraprotein electron transfer between flavin and Fe/S centers, (ii) the role of dynamics in interprotein electron transfer and (hi) quantum meehanieal mechanisms for the cleavage of substrate C-H bonds and the subsequent transfer of reducing equivalents to flavin redox centers. Brief mention is made of early structural and cofactor analyses for this redox system, but more detailed accounts of this work can be found in earlier reviews on the subjeet (e.g. Steenkamp and Mathews, 1992). 

Dobbek, H., Gremer, L., Meyer, O., and Huber, R., 1999, Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine, Proc. Nad. Acad. Sci. (USA) 96 8884118889. 

The catalytic cycle of each flavoenzyme consists of two distinct processes, the acceptance of redox equivalents from a substrate and the transfer of these equivalents to an acceptor. Accordingly, the catalyzed reactions consist of two half-reactions a reductive half-reaction in which the flavin is reduced and an oxidative half-reaction, in which the reduced flavin is reoxidized. This review summarizes the chemistry of simple flavoprotein reductases, dehydrogenases, (di)thiol oxidoreductases, oxidases, and monooxygenases (Table 1) (5 0) This grouping provides a good appreciation about what type of common mechanisms can be distinguished and what type of substrates can be converted. Information on the chemistry of complex flavoenzymes can be found in the Further Reading section. 

Although H-transfer in flavoprotein amine dehydrogenases has been shown to occur by tunneling, the mechanisms of amine oxidation by flavoproteins remain controversial. Over the years mechanisms involving the following have been con-... 

The enzyme D-lactate dehydrogenase from Megasphaera elsdenii catalyzes the oxidation of D-lactate to pyruvate, with an electron-transferring flavoprotein serving as the ultimate oxidant. Its reaction is similar to the first step of the lactate oxidase reaction, but the two enzymes use enantiomeric substrates, leading to the proposal that the two enzymes utilize similar mechanisms but bind their substrates in opposite orientations (Ghisla et al., 1976). Incubation of o-lactate dehydrogenase with d-13 leads to enzyme inactivation with a partition ratio of 5 (Olson et al., 1979). A novel pink chromophore formed concomitantly... 

A significant advance in understanding of the mechanism of the dihydrolipoic dehydrogenase reaction resulted from the discovery by Massey (1958) that the classic flavoprotein first isolated by Straub (1939), and widely known as Straub s diaphorase, behaves as a powerful dihydrolipoic dehydrogenase. Diaphorase activity was measured with ferricyanide as electron acceptor, Eq. (27), and dihydrolipoic dehydrogenase activity by... 

Fig. 37. Mechanism for oxidation of substrate (SHi) by flavoprotein (36). Y represents the yellow enzyme, fatty acyl CoA dehydrogenase FAD and FADH2 represent the oxidized and reduced forms, respectively, of enzyme-bound flavin and S and SHt represent the oxidized and reduced forms of substrate.

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