Oxidation coenzymes

As its name implies, the citric acid cycle is a closed loop of reactions in which the product of the hnal step (oxaloacetate) is a reactant in the first step. The intermediates are constantly regenerated and flow continuously through the cycle, which operates as long as the oxidizing coenzymes NAD+ and FAD are available. To meet this condition, the reduced coenzymes NADH and FADH2 must be reoxidized via the electron-transport chain, which in turn relies on oxygen as the ultimate electron acceptor. Thus, the cycle is dependent on the availability of oxygen and on the operation of the electron-transport chain. 

This sequence of reactions, namely oxidation of CH2-CH2 to CH=CH, then hydration to CH2-CHOH, followed by oxidation to CH2-CO, is a sequence we shall meet again in the -oxidation of fatty acids (see Section 15.4.1). The first oxidation utilizes FAD as coenzyme, the second NAD+. In both cases, participation of the oxidative phosphorylation system allows regeneration of the oxidized coenzyme and the subsequent generation of energy in the form of ATP. 

Oxidative coenzymes with structures of precisely determined oxidation-reduction potential. Examples are NAD+, NADP+, FAD, and lipoic acid. They serve as carriers of hydrogen atoms or of... 

When trying to determine the 3D structure of binary and ternary complexes of Drosophila alcohol dehydrogenase (DADH), researchers initial attempts to soak apo-form crystals with the oxidized coenzyme (NAD+) failed. The crystals cracked after several hours and became unusable. This suggested that the coenzyme, upon binding to the enzyme, induced a conformational change that seriously affected the crystal packing. The same phenomenon prevented solution of the HL-ADH 3D structure for many years. 

Figure 6. Sedimentation equilibrium of oxidized coenzyme A. Speed, 40,000 rpm temperature, 20.0° wavelength, 265 nm solvent, 0.05M potassium phosphate, pH 6.8.

This has been one of the most controversial areas of bioenergetics and is concerned with the role of coenzyme Q. The simplest view of the role of this coenzyme is that it acts as a mobile (2H+ + 2e ) carrier, linking complexes I and II with complex III. However, coenzyme Q may be involved in (H+ + e ) transfer within complex III. One model for this is the proton-motive Q cycle (Fig. 14-6), developed by Mitchell in 1975. This model satisfies prediction (2) of Example 14.10, in that coenzyme Q acts as an (H+ +e ) carrier in two loops. In this model, reduced coenzyme Q (QH2) is linked to oxidized coenzyme Q (Q) via the free-radical semiquinone (QH-) This model provides an explanation for the H+/e stoichiometry. 

As shown in Figure 8.5, the oxidized coenzymes have a formal positive charge, and are represented as NAD+ and NADP+, whereas the reduced forms, carrying two electrons and one proton (and associated with an additional proton), are represented as NADH and NADPH. The two-electron reduction of NAD(P)+ proceeds by way of a hydride (H ) ion transfer to carbon-4 of the nicotinamide ring. 

Use of NAD(P) in Enzyme Assays The reduced coenzymes have an absorption maximum at 340 nm, whereas the oxidized coenzymes do not. This is widely exploited to provide sensitive and specific methods for determining a variety of analytes using purified NAD (P)-linked enzymes and following the change in absorption at 340 nm as the coenzyme is either reduced or oxidized by the substrate. 

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