NAD reduction

Succinate-linked NAD reduction by R. rubrum chromatophores can be driven by PPj [9,10,88] according to the following equations  

This reaction can also be driven by light [9,10,88] or ATP [9,10,89]. The rates of the ATP and PPj-driven NAD reduction are about 20-30 and 6-12% of the light-driven reduction, respectively. The addition of both PPj and ATP causes a synergistic stimulation. Oligomycin inhibits the ATP-driven reaction but stimulates the PPj- as well as the light-driven reactions with 60-70 and 20-80%, respectively. The PPj-driven reaction is inhibited by uncouplers but not by electron-transport inhibitors. 

Chromatophores from R. rubrum can utilize the energy generated by the hydrolysis of PPj to drive ATP synthesis in the presence of P and ADP [11,12] according to the following reactions  

The opposite effects are observed with the succinate-linked NAD reduction. The NAD reduction is inhibited over 50% by PPj-driven ATP formation, but ATP formation is not inhibited at all by NAD reduction. NAD reduction apparently requires a higher level of than does the transhydrogenase. Thus, competing 

Once EPPj is formed, it is more likely to be cleaved than to appear as medium PR . If this reaction mechanism is true also for the membrane-bound PRase from R. rubrum the contribution of a membrane potential for the synthesis of PR should be on the release of the product RRj from the enzyme. 

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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. 

Recently, the use of pentamethylcyclopentadienyl(l,10-phenanthrohne-5,6-di-one)chloro rhodium(III) hexafluorophosphate [(Cp )Rhm(phend)Cl]PF6, 11 (Fig. 43.4) has been reported for the electrochemical NAD+ reduction. TONs between 7 and 453 h-1 have been achieved by varying pH, temperature and the complex concentrations [44]. This study reveals only preliminary results, so the mechanism of cofactor reduction is not explained however, due to the structural... 

For enzymatic reductions with NAD(P)H-dependent enzymes, the electrochemical regeneration of NAD(P)H always has to be performed by indirect electrochemical methods. Direct electrochemical reduction, which requires high overpotentials, in all cases leads to varying amounts of enzymatically inactive NAD-dimers generated due to the one-electron transfer reaction. One rather complex attempt to circumvent this problem is the combination of the NAD+ reduction by electrogenerated and regenerated potassium amalgam with the electrochemical reoxidation of the enzymatically inactive species, mainly NAD dimers, back to NAD+ [51]. If one-electron... 

Assay of bile acids was an essential tool for the early investigation of the enterohepatic circulation, and proved a focus of attention with the belief that serum bile-acid concentrations would provide a sensitive diagnostic test for liver disease. There are three fundamental assay types, based on enzymatic oxidation of a hydroxyl with linked NAD reduction, chromatographic separations and quantitation, encompassing both gas-liquid and high-performance liquid chromatography, and radioimmunoassay assays. 

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.

Protein fraction Ferredoxin fraction NAD reduction (nmol min mg protein" ) Total protein (mg) Ferredoxin protein (mg)... 

The first step in reaction scheme (4), believed to be the rate limiting step (43), produces a cation radical. The E0,pH7 of the NADH +/NADH couple in aqueous solutions has been estimated to 0.69 V (48,53), 0.72V (49), and 0.78 V vs SCE (52) by the use of one electron acceptors such as ferrocenes or ferricyanide. The deprotonation in the second step should be considered as irreversible due to the estimated low pK -4 (57)), of the cation radical, having a fast deprotonation rate, kH+> 106 s"1 (50). The second electron transfer in scheme (4) is the equivalent of the first step in the NAD+ reduction discussed above. It is depicted reversible to stress the chemical reversibility of this reaction, although it now takes place at a large positive working potential and therefore from an electrochemical point of view is irreversible. 

The pH optimum of the pig heart lipoamide dehydrogenase in the direction of NAD reduction by dihydrolipoamide is 7.9 (4). In the direction of NADH oxidation by lipoamide the pH optimum is 6.5 (4). In this latter direction there is an absolute requirement for NAD at the pH optimum (S7), but this requirement disappears as the pH is raised (116). It is therefore crucial to be aware of the pH of the measiu ements in comparing kinetic data. 

The kinetics of the yeast lipoamide dehydrogenase in the direction of NAD+ reduction indicate a bi-bi ping-pong mechanism is operative in this species also (117). If the enzyme from yeast indeed proves to have a tighter EHa-NADH complex than does the mammalian enzyme, product inhibitions studies should show impressive dependence on the fixed substrate (9S). 

Thus far a modifier role for NAD+ has been discussed only in the direction of NADH oxidation. Recent studies suggest that NAD may also have a double role in the direction of NAD+ reduction by dihydrolipo-... 

Ethanol production is essentially redox neutral however metabolism associated with biomass production generates nett NADH, which is oxidised largely by glycerol production. Other important NADH oxidising reactions with flavour implications are the production of 2,3-butanediol, L-malic acid and succinic add. When glycerol production is stimulated by non-growth associated reactions (i.e. osmotic stress) NAD+ reduction occurs by other reactions including the oxidation of acetaldehyde to acetic acid... 

An example of the use of a highly specialised cell type to study targeted toxic effects on the cellular metabolism is the recently developed boar spermatozoon motility inhibition test (Andersson et al., 1998). The motility of a spermatozoon depends on the integrity of mitochondrial functions, and thus the action of toxins affecting the energy metabolism is very rapidly detected as reduction of motility. Other end points that can be measured are plasma membrane integrity, astrodome function, and total cellular ATP and NAD reduction. This test has been particularly useful in the detection of certain types of bacterial toxins from various enviromnental and food sources. 

Those photosynthetic eubacteria with RC-2 centers (filamentous and purple bacteria) reduce NAD" for CO2 fixation by reverse electron flow from the quinone pool, whereas the green sulfur bacteria (RC-1 center) reduce ferredoxin and NAD directly from the secondary acceptor (Fe-S center) of the RC. In both cases an external reductant such as H2S is required. The mechanism of NAD reduction in the gram-positive line has not yet been investigated, but H. chlorum is a het-erotroph rather than an autotroph, and may not need to fix CO2. 

NAD" photoreduction in chromatophores isolated from several purple non-sulfur bacteria [41,48-50] and from the purple sulfur bacterium Chromatium vinosum [51] but did not inhibit ATP-driven NAD reduction in the dark. 

Determination of ethanol concentration is widely used in the wine, beer, and distilled beverage industries as well as in tissues. Ethanol is readily determined by the use of alcohol dehydrogenase by measuring the rate or extent of NAD reduction spectrophotometrically at 340 nm. 

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