Reduction of NADP

Fig. 5.2. The photosynthetic membrane of a green sulfur bacterium. The light-activated bacte-riochlorophyll molecule sends an electron through the electron-transport chain (as in respiration) creating a proton gradient and ATP synthesis. The electron eventually returns to the bacteri-ochlorophyll (cyclic photophosphorylation). If electrons are needed for C02 reduction (via reduction of NADP+), an external electron donor is required (sulfide that is oxidised to elemental sulfur). Note the use of Mg and Fe.

The enzyme ferridoxin (Fd) NADP + oxidoreductase accepts the electron from Fd, one at a time, as it proceeds from its oxidized form through a semiquinone intermediate to its fully reduced form. The enzyme then transfers a hydride ion to NADP converting to its reduced state NADPH. Uptake of a proton in the reduction of NADP+ further contributes to the proton gradient across the thylakoid membrane driving ATP synthesis. 

The reaction-center proteins for Photosystems I and II are labeled I and II, respectively. Key Z, the watersplitting enzyme which contains Mn P680 and Qu the primary donor and acceptor species in the reaction-center protein of Photosystem II Qi and Qt, probably plastoquinone molecules PQ, 6-8 plastoquinone molecules that mediate electron and proton transfer across the membrane from outside to inside Fe-S (an iron-sulfur protein), cytochrome f, and PC (plastocyanin), electron carrier proteins between Photosystems II and I P700 and Au the primary donor and acceptor species of the Photosystem I reaction-center protein At, Fe-S a and FeSB, membrane-bound secondary acceptors which are probably Fe-S centers Fd, soluble ferredoxin Fe-S protein and fp, is the flavoprotein that functions as the enzyme that carries out the reduction of NADP+ to NADPH. 

In 1931 Warburg and Christian (W5) detected an enzyme catalyzing the reduction of NADP by G-6-P, which he called Zwischenferment G-6-P + NADP+ 6-PG + NADPH + H+ (3)... 

Another enzyme used for the measurement of glucose is hexokinase (EC 2.7.1.1) which catalyses the phosphorylation of glucose to produce glucose-6-phosphate with adenosine triphosphate as the phosphate donor and magnesium ions as an activator. The rate of formation of glucose-6-phosphate can be linked to the reduction of NADP by the enzyme glucose-6-phosphate dehydrogenase (EC 1.1.1.49). This indicator reaction can be monitored spectrophotometrically at 340 nm or fluorimetrically ... 

The final reactions to be considered in the metabolism of ethanol in the liver are those involved in reoxidation of cytosolic NADH and in the reduction of NADP. The latter is achieved by the pentose phosphate pathway which has a high capacity in the liver (Chapter 6). The cytosolic NADH is reoxidised mainly by the mitochondrial electron transfer system, which means that substrate shuttles must be used to transport the hydrogen atoms into the mitochondria. The malate/aspartate is the main shuttle involved. Under some conditions, the rate of transfer of hydrogen atoms by the shuttle is less than the rate of NADH generation so that the redox state in the cytosolic compartment of the liver becomes highly reduced and the concentration of NAD severely decreased. This limits the rate of ethanol oxidation by alcohol dehydrogenase. 

The light reactions in photosynthesis bring about two strongly endergonic reactions—the reduction of NADP to NADPH+H and ATP synthesis (see p. 122). The chemical energy needed for this is produced from radiant energy by two photosystems. 

This unique redox catalyst links the oxidation of H2 or of formate to the reduction of NADP+229 and also serves as the reductant in the final step of methane biosynthesis (see Section E) 228 It resembles NAD+ in having a redox potential of about -0.345 volts and the tendency to be only a two-electron donor. More recently free 8-hydroxy-7,8-didemethyl-5-deazaribo-flavin has been identified as an essential light-absorbing chromophore in DNA photolyase of Methanobacterium, other bacteria, and eukaryotic algae.230 Roseoflavin is not a coenzyme but an antibiotic from Streptomyces davawensisP1 Many synthetic flavins have been used in studies of mechanisms and for NMR232 and other forms of spectroscopy. 

Figure 20-7 Simplified representation of the photoreactions in photosynthesis. The oxidation of water is linked to the reduction of NADP by an electron-transport chain (dashed line) that is coupled to ATP formation (photophosphorylation).

To understand why isocitrate dehydrogenase is so intensely regulated we must consider reactions beyond the TCA cycle, and indeed beyond the mitochondrion (fig. 13.15). Of the two compounds citrate and isocitrate, only citrate is transported across the barrier imposed by the mitochondrial membrane. Citrate that passes from the mitochondrion to the cytosol plays a major role in biosynthesis, both because of its immediate regulatory properties and because of the chain of covalent reactions it initiates. In the cytosol citrate undergoes a cleavage reaction in which acetyl-CoA is produced. The other cleavage product, oxaloacetate, can be utilized directly in various biosynthetic reactions or it can be converted to malate. The malate so formed can be returned to the mitochondrion, or it can be converted in the cytosol to pyruvate, which also results in the reduction of NADP+ to NADPH. The pyruvate is either utilized directly in biosynthetic processes, or like malate, can return to the mitochondrion. 

Rat liver mitochondria were isolated as described (4). The initial rate of ATP synthesis associated with the oxidation of succinate was followed by monitoring fluorometrically the ATP-linked NADPH production in the presence of hexokinase and glucose-6-phosphate dehydrogenase (10). Control experiments showed no interference from unexpected reduction of NADP+ or from electron backflow. Possible ATP formation via mitochondrial adenylate... 

The light-driven, energy- and electron-transfer processes trigger reactions that finally lead to the oxidation of water, the reduction of NADP+, and the build-up of a proton gradient across the photosynthetic membrane to produce ATP These processes are catalyzed by two membrane-embedded pigment-protein complexes, called... 

For the asymmetric reduction of ketone and aldehyde derivates, two electrochemical reduction systems using ADH as catalyst were examined (Fig. 22) [108]. In system A, the reduced coenzymes are regenerated using either FNR for NADPH or DP for NADH. Methyl viologen serves as electron mediator between the electrode and FNR/DP. System B contains ADH as sole enzyme, which catalyzes both reduction of substrates and regeneration of cofactors. Phenylethanol is oxidized by ADH accompanied by reduction of NADP+ to NADPH and its oxidation product acetophenone is reduced electrochemically at a glassy carbon cathode. 

The photochemical reaction of photosynthesis involves the removal of an electron from an excited state of the special chlorophyll that acts as an excitation trap. The movement of the electron from this trap chi to an acceptor begins a series of electron transfers that can ultimately lead to the reduction of NADP+. The oxidized trap chi, which has lost an electron, can accept another electron from some donor, as in the steps leading to O2 evolution. Coupled to the electron transfer reactions in chloroplasts is the formation of ATP, a process known as photophosphorylation. In this section we will consider some of the components of chloroplasts involved in accepting and donating electrons a discussion of the energetics of such processes will follow in Chapter 6 (Section 6.3). 

Reduction of NAD+ by NADPH or reduction of NADP+ by NADH cannot be assayed directly since the spectral differences between NADH and NADPH or between NAD and NADP are negligible. Either reaction may be measured by removing aliquots from the reaction mixture... 

At neutral pH, the maximal initial velocities of the two directions of the non-energy-linked transhydrogenase reaction differ by a factor of about five, the reduction of NADP being the slower reaction (SO, 69, 68, 71, 127). Reduction of NADP by NADH is maximally active at about pH 5.5., whereas reduction of NAD by NADPH shows a pH optimum at about 7.0 (SO, 67, 71, 72 see also S2). When the reduction of NADP by NADH approaches equilibrium, the rate constant of the reaction is increased (67), indicating an activation of the transhydrogenase that is related to the accumulation of the products NAD and NADPH. It has been proposed (67, 69, 71) that this activation may involve a conversion of the enzyme from an inactive to an active conformational state, similar to that proposed to occur upon energization (see below). 

The lack of reactivity of the semiquinone per se with either thioredoxin or NADPH shows that it cannot be involved in catalysis. The rapid production of semiquinone by irradiation of partially reduced enzyme is a light-activated disproportionation since it is totally dependent upon the presence of some oxidized enzyme. Enzyme fully reduced by dithionite forms no semiquinone, while enzyme partially reduced by dithionite rapidly forms semiquinone upon irradiation. Furthermore, the light-activated disproportionation of enzyme first reduced with NADPH results in the reduction of NADP. Thus, FAD catalyzes the disproportionation in keeping with the known photosensitizing nature of free flavins. This reaction is reversed slowly (half-time ca. 150 min 25°) in the dark. The semiquinone is rapidly reoxidized by oxygen to yield an enzyme with unaltered spectral and catalytic properties (58). Similar reactions have been very briefly reported for lipoamide dehydrogenase the dark reverse reaction is comparatively rapid, being complete in 30 min (16S). 

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