NADH oxidation

Analytical models of modified electrodes for NADH oxidation 

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ELECTROPOLYMERIZED FLAVINS AND AZINES AS ELECTROCATALYSTS FOR NADH OXIDATION... 

Engelhardt s experiments in 1930 led to the notion that ATP is synthesized as the result of electron transport, and, by 1940, Severo Ochoa had carried out a measurement of the P/O ratio, the number of molecules of ATP generated per atom of oxygen consumed in the electron transport chain. Because two electrons are transferred down the chain per oxygen atom reduced, the P/O ratio also reflects the ratio of ATPs synthesized per pair of electrons consumed. After many tedious and careful measurements, scientists decided that the P/O ratio was 3 for NADH oxidation and 2 for succinate (that is, [FADHg]) oxidation. Electron flow and ATP synthesis are very tightly coupled in the sense that, in normal mitochondria, neither occurs without the other. 

Oxidation of 2 molecules each of isocitrate, n-ketoglutarate, and malate yields 6 NADH Oxidation of 2 molecules of succinate yields 2 [FADHg] Oxidative phosphorylation (mitochondria) 2 NADH from glycolysis yield 1.5 ATP each if NADH is oxidized by glycerol-phosphate shuttle 2.5 ATP by malate-aspartate shuttle + 3 + 5... 

Barker CD, Reda T, Hirst J. 2007. The flavoprotein suhcomplex of complex I (NADH ubiquinone oxidoreductase) from bovine heart mitochondria Insights into the mechanisms of NADH oxidation and NAD reduction from protein film voltammetry. Biochemistry 46 3454-3464. 

Maas FM, De Kok LJ. 1988. In vitro NADH oxidation as an early indicator for growth reduction of spinach exposed to hydrogen sulfide in the ambient air. Plant Cell Physiol 29 523-526. 

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],... 

A. Ciszewski and G. Milczarek, Electrocatalysis of NADH oxidation with an electropolymerized film of l,4-bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane. Anal. Chem. 72, 3203-3209 (2000). 

N. Mano and A. Kuhn, Immobilized nitro-fluorenone derivatives as electrocatalysts for NADH oxidation. J. Electroanal. Chem. 477, 79-88 (1999). 

A.A. Karyakin, E.E. Karyakina, W. Schuhmann, and H.L. Schmidt, Electropolymerized azines Part II. In search of the best electrocatalyst of NADH oxidation. Electrocatalysis 11, 553-557 (1999). 

Rotenone inhibits the transfer of electrons from NADH into the electron transport chain. The oxidation of substrates that generate NADH is, therefore, blocked. However, substrates that are oxidized to generate FADH2 (such as succinate or a-glycerol phosphate) can still be oxidized and still generate ATP. Because NADH oxidation is blocked, the NADH pool becomes more reduced in the presence of rotenone since there s nowhere to transfer the electrons. 

At the beginning only XO and not XDH was considered as a superoxide producer. For example, in 1985 McCord [19] suggested that the conversion of XDH into XO is responsible for an increase in superoxide production in postischemic reperfusion injury. However, it has later been shown [20,21] that XDH itself is a producer of superoxide although not so effective as XO. Moreover, the efficiency of superoxide production differs for different types of the enzyme. Thus, 2.8 to 3.0 mol of superoxide were produced by chicken liver XDH, while superoxide production by bovine milk XDH was insignificant [21]. Sanders et al. [22] found that NADH oxidation by human milk and by bovine milk XDHs catalyzed superoxide production more rapidly than XO this process was inhibited by NAD and diphenyleneiodo-nium but not by the established XO inhibitors allopurinol and oxypurinol. 

GLYCEROL PHOSPHATE DEHYDROGENASE IS AN NADH OXIDIZING ENZYME RELATED TO GLYCOLYSIS 541... 

The amount of NADH oxidized and hence (he amount of amino acid is proportional to the absorbance change. 

Cytoplasmic NADH oxidized using the malate shuttle produces a mitochondrial NADH and yields approximately 3 ATP by oxidative phosphorylation. 

Cytoplasmic NADH oxidized by the glycerol phosphate shuttle produces a mitochondrial FADHj and yields approximately 2 ATP by oxidative phosphorylation. 

NADH oxidation via complex I takes place on the inside of the membrane—i. e., in the matrix space, where the tricarboxylic acid cycle and 3-oxidation (the most important sources of NADH) are also located. O2 reduction and ATP formation also take place in the matrix. 

The inner membrane itself plays an important part in oxidative phosphorylation. As it is impermeable to protons, the respiratory chain—which pumps protons from the matrix into the intermembrane space via complexes 1, 111, and IV—establishes a proton gradient across the inner membrane, in which the chemical energy released during NADH oxidation is conserved (see p. 126). ATP synthase then uses the energy stored in the gradient to form ATP from ADP and inorganic phosphate. Several of the transport systems are also dependent on the H"" gradient. 

Figure 5. Ultraviolet spectrum of NAD+ and NADH. Note that the absorption band centered at 340 nm serves as a valuable way to assay many dehydrogenases as well as other enzymes that form products that can be coupled to NAD+ reduction or NADH oxidation.

NADH/NADPH SURFACE FLUORESCENCE IN LIVING TISSUES NADH oxidation,... 

In mitochondria, the allelochemlcals acted primarily as electron transport inhibitors. Malate oxidation was more sensitive than either succinate or NADH oxidation. No evidence for interaction with a specific membrane complex was obtained. Instead, Inhibition of substrate oxidation seems to result from alterations and perturbations produced in the inner membrane as reflected in interference with the behavior of transport processes. The compounds did not act as uncouplers or directly inhibit ATP synthesis. However, naringenin, some of the flavones, and the cinnamic acids dj inhibit the hydrolysis of ATP catalyzed by mitochondrial Mg -ATPase. 

The kinetics of NADH oxidation by three vanado-tungstodiphosphoric species were studied at pH = 7 by the stopped flow technique [82], giving values in complete agreement with electrochemistry results to be discussed in the following. 

The ADP produced by the hydrolysis of ATP is continuously used up by added purified pyruvate kinase, which in the presence of phosphoenol pyruvate produces pyruvate and ATP (Fig. 3.8.6). Pyruvate is then utilized by added lactate dehydrogenase, which in the presence of NADH produces lactate and NAD+. Complex V activity is estimated from the rate of NADH oxidation at 340 nm (e 4870-M 1-cm 1 isosbestic point 380 nm), after subtracting the oligomycin-resistant activity. It should be kept in mind that oligomicyn sensitivity requires the preserved attachment of the Fr component of the enzyme to the membranous F0 component. The attachment is readily lost upon freeze-thaw cycles. Consequently, it is reasonable to measure the activity on fresh material only. 

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