NAD+/NADH redox couple

Reaction (1) involves a stereospecific net transfer of a hydride ion between a substrate and the C(4) of the pyridine ring of the coenzyme and an exchange of a proton with the medium 14). The generally accepted formal potential, E° of the NAD+/NADH redox couple is -560 mV vs SCE (pH 7, 25°C) 15). This value is obtained from equilibrium constants of dehydrogenase catalyzed reactions and thermal data. For most systems the equilibrium of reaction (1) favors the substrate rather than the product side. The reason for this is the low oxidizing power of NAD+, which is reflected by the low value of E° ... 

The formal potential of the NAD+/NADH redox couple is -0.56 V vs. SCE at pH 7 [15, 17]. However, at platinum and glassy carbon electrodes NADH, oxidation occurs at 0.7 V and 0.6 V vs. SCE, respectively [18]. From these oxidation potentials, it is clear that the direct electrochemical oxidation of NADH requires a substantial overpotential. In nature, NADH oxidation is thought to occur by a one-step hydride transfer. However, on bare electrodes the reaction has been shown to occur via a different and higher energy pathway which produces NAD radicals as intermediates. 

Why is it important for the value of AE q for the NAD" NADH redox couple to be less negative than those for the redox couples of oxidizable compounds that are components of the glycolytic pathway and the citric acid cycle ... 

The electrochemical sensing of NADH is of great interest in the development of a dehydrogenase-based amperometric biosensor owing to the ubiquitous use of NADH as a cofactor for over 300 enzymes and in the fine chemicals industry using NAD -dependent biocatalysts [106]. The oxidation of NADH at bare and modified electrodes has been well studied and the oxidation process is dependent on the nature of the electrode used. The direct electrochemical oxidation of NADH at the bare electrode, irrespective of its nature, requires a high overpotential, despite the formal potential of the NAD /NADH redox couple at pH 7, which is reported to be... 

The generally accepted formal potential, E°, of the NAD+/NADH redox couple at pH 7.0 (25 °C) is —315 mV versus normal hydrogen electrode (NHE)(—560 mV vs. saturated calomel electrode (SCE) [23, 24). From thermal data and the equilibrium constants of the ethanol/acetaldehyde and 2-propanol/acetone reactions catalyzed by alcohol dehydrogenase, a value of —320 mV was calculated, which was later recalculated to be —315 5 mV versus NHE by Clark [23]. Through direct potentiometric titrations using several different mediators and xanthine oxidase as catalyst, Rodkey [25, 26] obtained an E° value of —311 mV versus NHE (25 °C) and a temperature variation of the E° of —1.31 mV/ C in the range of 20 to 40 °C. A variation of the E° with pH of —30.3 mV/pH (30 C) was found, which... 

Whereas mitochondrial oxidation utilizes NAD" and flavoproteins coupled to the oxidative phosphorylation system, the glyoxysomal system differs in two m jor respects (Fig. 5). First, the flavoprotein reduced in the acyl-CoA dehydrogenase step is oxidized directly by molecular oxygen with the formation of HjOj, which is then degraded by catalase second, NADHj produced in the oxidation of 2-hydroxyacyl-CoA is not reoxidized in the glyoxy-somes thus an external NAD -NADH redox system is required. 

On the basis of recent findings by Tunon-Blanco and coworkers [139], one could also speculate that the adenine moiety of adsorbed NAD" " may undergo an oxidative reaction at high anodic potentials, forming a strongly mediating functionality on the electrode surface and thus facilitating the oxidation of NADH at potentials below the E° of the NADH /NADH redox couple, (Fig. 6). 

The half-reactions and reduction potentials in Table 21.1 can be used to analyze energy changes in redox reactions. The oxidation of NADH to NAD can be coupled with the reduction of a-ketoglutarate to isocitrate ... 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

It is important not to confuse the reactions of Eq. 17-42 as they occur in an aerobic cell with the tightly coupled pair of redox reactions in the homolactate fermentation (Fig. 10-3 Eq. 17-19). Tire reactions of steps a and c of Eq. 17-42 are essentially at equilibrium, but the reaction of step b may be relatively slow. Furthermore, pyruvate is utilized in many other metabolic pathways and ATP is hydrolyzed and converted to ADP through innumerable processes taking place within the cell. Reduced NAD does not cycle between the two enzymes in a stoichiometric way and the "reducing equivalents" of NADH formed are, in large measure, transferred to the mitochondria. The proper view of the reactions of Eq. 17-42 is that the redox pairs represent a kind of redox buffer system that poises the NAD+/NADH couple at a ratio appropriate for its metabolic function. 

Apparatus for measuring the difference between the E° values of two redox couples. The cell on the left contains equimolar concentrations of NADH and NAD + that on the right, equimolar concentrations of FMNH2 and FMN. If both solutions are at pH 7, the voltmeter senses the difference between the two E° values (0.10 V, negative on the left, in this example). To determine the E° of one of the redox couples, the other couple is replaced by a standard redox couple. 

Another class of energy storage compounds consists of redox couples such as NADP+-NADPH (Table 6-1). The reduced form, NADPH, is produced by noncyclic electron flow in chloroplasts (Chapter 5, Section 5.5C). Photosynthesis in bacteria makes use of a different redox couple, NAD+-NADH. The reduced member of this latter couple also causes an... 

Fig. 3.1. A, The respiratory chain. Q and c stand for ubiquinone and cytochrome c, respectively. Auxiliary enzymes that reduce ubiquinone include succinate dehydrogenase (Complex II), a-glycerophosphate dehydrogenase and the electron-transferring flavoprotein (ETF) of fatty acid oxidation. Auxiliary enzymes that reduce cytochrome c include sulphite oxidase. B, Thermodynamic view of the respiratory chain in the resting state (State 4). Approximate values are calculated according to the Nernst equation using oxidoreduction states from work by Muraoka and Slater, (NAD, Q, cytochromes c c, and a oxidation of succinate [6]), and Wilson and Erecinska (b-562 and b-566 [7]). The NAD, Q, cytochrome b-562 and oxygen/water couples are assumed to equilibrate protonically with the M phase at pH 8 [7,8]. E j (A ,/ApH) for NAD, Q, 6-562, and oxygen/water are taken as —320 mV ( — 30 mV/pH), 66 mV (- 60 mV/pH), 40 mV (- 60 mV/pH), and 800 mV (- 60 mV/pH) [7-10]. FMN and the FeS centres of Complex I (except N-2) are assumed to be in redox equilibrium with the NAD/NADH couple, FeS(N-2) with ubiquinone [11], and cytochrome c, and the Rieske FeS centre with cytochrome c [10]. The position of cytochrome a in the figure stems from its redox state [6] and its apparent effective E -, 285 mV in...

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