The oxidation of NADH

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

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 ... 

Consider the oxidation of NADH by molecular oxygen as carried out via the electron transport pathway... 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Ferricyanide has been employed as an electron acceptor for lactate in place of NAD Alternatively, diaphorase can be used in conjunction with ferricyanide Bindschedler s Green ferricenium or dichlorophenylindophenol to catalyze the oxidation of NADH while providing a more easily monitored species than NADH itself. 

The famous model for the oxidation of NADH coupled with the proton transport in a mitochondria is the Q cycle [53], as follows NADH in the aqueous solution (matrix) is oxidized to NAD by Q in the membrane producing hydroquinone (QH2). 

Taking into account the results obtained by polarography as well as controlled potential electrolysis, the reaction which proceeded in Range A and gave the polarographic wave was estimated to be composed of two-electron oxidation of NADH and one-electron reduction of CQ at the W/DCE interface. The oxidation of NADH is accompanied by the dissociation of one H in W. 

Simon et al. [92] investigated a biocatalytic anode based on lactate oxidation by lactate dehydrogenase (LDH). The anodic current is generated by the oxidation of NADH (produced by NAD+ and substrate) while LDH catalyzes the electro-oxidation of lactate into pyruvate. As previously mentioned, the oxidation of NADH at bare electrodes requires a large overpotential, so these authors used poly(aniline) films doped with polyanions to catalyze NADH oxidation. Subsequent research by this group focused on targeting mutants of LDH that are amenable to immobilization on the polyaniline surface [93],... 

The P/O ratio is the number of ATPs made for each O atom consumed by mitochondrial respiration. The P stands for high-energy phosphate equivalents, and the O actually stands for the number of I 02 s that are consumed by the electron transport chain. The full reduction of 02 to 2 H20 takes 4 electrons. Therefore, 2 electrons reduce of an 02. The oxidation of NADH to NAD and the oxidation of FADH2 to FAD are both 2-electron oxidations. O can be read as the transfer of 2 electrons. It s not quite as obscure as it sounds.2... 

In the presence of the inhibitor rotenone (to prevent the oxidation of NADH by the electron transport chain), succinate can be metabolized only to fumarate, producing an FADH2 in the process. 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Electrons enter the respiratory chain in various different ways. In the oxidation of NADH+H" by complex I, electrons pass via FMN and Fe/S clusters to ubiquinone (Q). Electrons arising during the oxidation of succinate, acyl CoA, and other substrates are passed to ubiquinone by succinate dehydrogenase or other mitochondrial dehydrogenases via en-... 

Examination of one real-life case may benefit the reader s understanding. Strittmatter studied the primary kinetic isotope effects arising in the NADH-dependent cytochrome bs reductase (EC 1.6.2.2). The oxidation of NADH and subsequent reduction of cytochrome bs is facilitated by the enzyme-bound FAD group, and the kinetics of the direct transfer of a hydrogen from the A-face (or pro-R) of NADH to the flavin can be monitored by the loss of the 340 run absorbance of the NADH s dihydropyridine ring. Using deuterated isotopic isomers of NADH and several related compounds, Strittmatter obtained the primary kinetic isotope effect data compiled in the table below. 

Figure 33 shows a representative example of the oxidation of NADH by POMs using cyclic voltammetry at a scan rate of 5 mV s k Other examples can be found in the original papers. Typically, the system 1-[P2VWi7062] /NADH is shown. The... 

Scheme 15 The initial one-electron rate-limiting step in the oxidation of NADH by the ROMs studied in this work (taken from Ref. 174).

Oxidative Phosphorylation. Oxidative phosphorylation, that is the production of ATP during the passage of electrons down the terminal electron transport chain, may be disrupted in two distinct ways. Compounds that divorce the process of electron transport and the phosphorylation of ADP are termed uncoupling agents. They permit NADH and succinate to be oxidised via the electron transport chain without the production of ATP and are lethal. Oxidative phosphorylation may also be inhibited directly, thus preventing the oxidation of NADH and succinate. Several products are available that exploit these modes of action. Characteristically, they have wide activity spectra that span major disciplines of pesticide use. 

Z F6P + ATP + 2 NADH + H+ <-> 2 glycerol-3-phosphate + ADP + 2 NAD+ where F6P is fructose-6-phosphate, FDP is fructose-1,6-diphosphate, DHA-P is dihydroxyacetone phosphate, TIM is triosephosphate isomerase, and GDH is glycerol-3-phosphate dehydrogenase. The oxidation of NADH is a measure of the 6-PFK activity and is determined photometrically (decrease of OD per minute) [4]. 

In a number of earlier studies the oxidation of NADH or NADPH was assayed under conditions (acid pH, Mn ) in which there was a large component of what appears to be a chain reaction propagated by free radicals Under these conditions it is difficult to be certain how much of the formation of O and of the oxidation of pyridine nucleotides was due to turnover of the enzyme and how much was due to the chain reaction. 

Redox pairs Oxidation (loss of electrons) of one compound is always accompanied by reduction (gain of electrons) of a second substance. For example, Figure 6.11 shows the oxidation of NADH to NAD+ accompanied by the reduction of FAD to FADH2. Such oxidation-reduction reactions can be written as the sum of two halfreactions an isolated oxidation reaction and a separate reduction reaction (see Figure 6.11). NAD+ and NADH form a redox pair, as do FAD and FADH2. Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair, and can be quantitatively specified by a constant, E (the standard reduction potential), with units in volts. 

Aerobic respiration can be subdivided into a number of distinct but coupled processes, such as the carbon flow pathways resulting in the production of carbon dioxide and the oxidation of NADH + H+ and FADH2 (flavin adenine dinucleotide) to water via the electron transport systems or the respiratory chain. 

Since two molecules of ATP are converted to ADP in the first part of the glycolysis process, there is a net gain of two molecules of ATP. The second part of the glycolysis process also yields two molecules of NADH + H+ per molecule of glucose. Subsequently, the energy-yielding conversion of the two molecules of ATP back to ADP and the oxidation of NADH,... 

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