Reduction of NAD

The citric acid cycle, a nine-step process, also diverts chemical energy to the production of ATP and the reduction of NAD and FAD. In each step of the citric acid cycle (also known as the Krebs cycle) a glucose metabolite is oxidized while one of the carrier molecules, NAD or FAD, is reduced. Enzymes, nature s chemical catalysts, do a remarkable job of coupling the oxidation and reduction reactions so that energy is transferred with great efficiency. 

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

Rhodium and ruthenium complexes have also been studied as effective catalysts. Rh(diphos)2Cl [diphos = l,2-bis(diphenyl-phosphino)ethane] catalyzed the electroreduction of C02 in acetonitrile solution.146 Formate was produced at current efficiencies of ca. 20-40% in dry acetonitrile at ca. -1.5 V (versus Ag wire). It was suggested that acetonitrile itself was the source of the hydrogen atom and that formation of the hydride HRh(diphos)2 as an active intermediate was involved. Rh(bpy)3Cl3, which had been used as a catalyst for the two-electron reduction of NAD+ (nicotinamide adenine dinucleotide) to NADH by Wienkamp and Steckhan,147 has also acted as a catalyst for C02 reduction in aqueous solutions (0.1 M TEAP) at -1.1 V versus SCE using Hg, Pb, In, graphite, and n-Ti02 electrodes.148 Formate was the main... 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

Fig. 2.2 Schematic diagram showing XOR-catalysed oxidation of hypoxanthine and xanthine (also most reducing substrates) at the molybdenum (Mo) site, and ofNADH at the FAD site. Reduction of NAD+ or of molecular oxygen takes place at FAD. Adapted from Harrison [73],...

The electrochemical activation of the catalyst must be possible at potentials less negative than — 0.9 V vs SCE since at more negative potentials the direct electrochemical reduction of NAD(P)+ will lead to NAD dimer formation. 

Determination of the Michaelis constant for the cofactor NAD (A m.NAo) was carried out by measuring the initial rate of the reduction of NAD as a function of its concentration, at a constant concentration of glucose. All solutions were prepared in 0.1 M phosphate buffer pH 7.55. 

Bacterial ferredoxins function primarily as electron carriers in ferredoxin-mediated oxidation reduction reactions. Some examples are reduction of NAD, NADP, FMN, FAD, sulfite and protons in anaerobic bacteria, CO -fixation cycles in photosynthetic bacteria, nitrogen fixation in anaerobic nitrogen fixing bacteria, and reductive carboxylation of substrates in fermentative bacteria. The roles of bacterial ferredoxins in these reactions have been summarized by Orme-Johnson (2), Buchanan and Arnon (3), and Mortenson and Nakos (31). 

Formation of 1,3-bisphosphoglycerate is coupled to reduction of NAD by transfer of two electrons and a proton to form NADH + H. ... 

A simplified picture of the electrochemical reduction of NAD+ (260) to NADH (262) and other products is illustrated (Scheme 185) (75JA5083). The first reduction of NAD+ to the radical (261) is pH independent, and this free radical rapidly dimerizes to (263) or (264). At more negative potential, NAD+ is reduced to a dihydropyridine by one-electron reduction of (261), but the dimer is not reduced at this potential. 

The acceptor of hydrogen in the glyceraldehyde 3-phosphate dehydrogenase reaction is NAD+ (see Fig. 13-15), bound to a Rossmann fold as shown in Figure 13-16. The reduction of NAD+ proceeds by the enzymatic transfer of a hydride ion ( H ) from the aldehyde group of glyceraldehyde 3-phosphate to the nicoti-... 

Alcohol dehydrogenase is present in many organisms that metabolize ethanol, including humans. In human liver it catalyzes the oxidation of ethanol, either ingested or produced by intestinal microorganisms, with the concomitant reduction of NAD+ to NADH. 

The reduction of NAD+ at a mercury electrode at —1.1 V versus SCE gives a 90% yield of dimers220 three stereoisomers of 4,4 -dimers were found to account for 90% of the dimer mixture, and three 4,6 -dimers were responsible for the remaining 10%. The dimers were separated, using reverse-phase HPLC and gel filtration on Sephadex G-15 no 6,6 -dimers were detected. Reduction at —1.8 V resulted in 50% 1,4-NADH, 30% 1,6-NADH, and 20% dimers. In other investigations221 the three stereoisomeric 4,4 -dimers have also been obtained as the main products. 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

The chemistry of the reduction of NAD+ has been solved most elegantly (Chapter 8, section Bi).2 Oxidation of the alcohol involves the removal of two hydrogen atoms. One is transferred directly to the 4 position of the nicotinamide ring of the NAD+, and the other is released as a proton (equation 16.1).3,4 It is generally thought that the hydrogen is transferred as a hydride ion H , but a radical intermediate cannot be ruled out. For convenience, we shall assume that the mechanism is the hydride transfer. 

Takahashi and co-workers (69,70,71) reported both cathodic and anodic photocurrents in addition to corresponding positive and negative photovoltages at solvent-evaporated films of a Chl-oxidant mixture and a Chl-reductant mixture, respectively, on platinum electrodes. Various redox species were examined, respectively, as a donor or acceptor added in an aqueous electrolyte (69). In a typical experiment (71), NAD and Fe(CN)g, each dissolved in a neutral electrolyte solution, were employed as an acceptor for a photocathode and a donor for a photoanode, respectively, and the photoreduction of NAD at a Chl-naphthoquinone-coated cathode and the photooxidation of Fe(CN)J at a Chl-anthrahydroquinone-coated anode were performed under either short circuit conditions or potentiostatic conditions. The reduction of NAD at the photocathode was demonstrated as a model for the photosynthetic system I. In their studies, the photoactive species was attributed to the composite of Chl-oxidant or -reductant (70). A p-type semiconductor model was proposed as the mechanism for photocurrent generation at the Chi photocathode (71). 

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