Pyridine nucleotide complexes

Reoxidation of the enzyme-pyridine nucleotide complex by ferricyanide takes place in two one-electron steps. The rate of the first step is too rapid to measure and the rate of the second step can be measured only if NADPH is used to prereduce the enzyme. In this case the complex with NADP+ is virtually nonexistent and the changes measured are those of uncomplexed enzyme. These observations demonstrate that a species is formed within the mixing time and that it decays rapidly (78 sec", 0°). Repetition at 10 nm intervals generates the spectrum of the intermediate which is that of the neutral semiquinone. The fact that both steps in the reoxidation of the enzyme-pyridine nucleotide complexes (i.e., when NADH is the reductant) are complete in less than 2 msec demonstrates that they meet the kinetic requirements of an intermediate, rapid formation and reoxidation 3S5). 

Reoxidation of the enzyme-pyridine nucleotide complexes by cjrto-chrome bs also takes place in two steps. The rate of the first step is again too fast to measure. The rate of the second step is markedly dependent upon the pyridine nucleotide involved being 190, 68, and 18 sec , 0° for NADH, AcPyADH, and NADPH, respectively S5B). 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

The chemistry of flavins is complex, a fact that is reflected in the uncertainity that has accompanied efforts to understand mechanisms. For flavoproteins at least four mechanistic possibilities must be considered.1533 233 (a) A reasonable hydride-transfer mechanism can be written for flavoprotein dehydrogenases (Eq. 15-23). The hydride ion is donated at N-5 and a proton is accepted at N-l. The oxidation of alcohols, amines, ketones, and reduced pyridine nucleotides can all be visualized in this way. Support for such a mechanism came from study of the nonenzymatic oxidation of NADH by flavins, a reaction that occurs at moderate speed in water at room temperature. A variety of flavins and dihydropyridine derivatives have been studied, and the electronic effects observed for the reaction are compatible with the hydride ion mecha-nism.234 236... 

Generally, the assimilatory nitrate and nitrite reductases are soluble enzymes that utilize reduced pyridine nucleotides or reduced ferrodoxin. In contrast, the dissimilatory nitrate reductases are membrane-bound terminal electron acceptors that are tightly linked to cytochrome by pigments. Such complexes allow one or more sites of energy conservation (ATP generation) coupled with electron transport. 

Frey PA (1987) Complex pyridine-dependent transformations. In Dolphin D, Poulson R, Avamovic O (eds) Pyridine nucleotide coenzymes Chemical biochemical, medical aspects Vol 2B. Wiley, New York, 462... 

This means that this dehydrogenase can form binary complexes with steroid substrates, binary complexes with pyridine nucleotide, and ternary complexes with both substrates. This behavior contrasts with that usually observed for NAD-linked dehydrogenases in which the ketone or aldehyde substrate can bind only to the NADH-enzyme binary complex but not to free enzyme (29). 

As is indicated in Table 5-3, P680, P70o> the cytochromes, plastocyanin, and ferredoxin accept or donate only one electron per molecule. These electrons interact with NADP+ and the plastoquinones, both of which transfer two electrons at a time. The two electrons that reduce plastoquinone come sequentially from the same Photosystem II these two electrons can reduce the two >-hemes in the Cyt b(f complex, or a >-heme and the Rieske Fe-S protein, before sequentially going to the /-heme. The enzyme ferre-doxin-NADP+ oxidoreductase matches the one-electron chemistry of ferredoxin to the two-electron chemistry of NADP. Both the pyridine nucleotides and the plastoquinones are considerably more numerous than are other molecules involved with photosynthetic electron flow (Table 5-3), which has important implications for the electron transfer reactions. Moreover, NADP+ is soluble in aqueous solutions and so can diffuse to the ferredoxin-NADP+ oxidoreductase, where two electrons are transferred to it to yield NADPH (besides NADP+ and NADPH, ferredoxin and plastocyanin are also soluble in aqueous solutions). 

Mitochondria contain ubiquinone (also known as coenzyme Q), which differs from plastoquinone A (Chapter 5, Section 5.5B) by two methoxy groups in place of the methyl groups on the ring, and 10 instead of 9 isoprene units in the side chain. A c-type cytochrome, referred to as Cyt Ci in animal mitochondria, intervenes just before Cyt c a h-type cytochrome occurring in plant mitochondria is involved with an electron transfer that bypasses cytochrome oxidase on the way to 02. The cytochrome oxidase complex contains two Cyt a plus two Cyt a3 molecules and copper on an equimolar basis with the hemes (see Fig. 5-16). Both the Fe of the heme of Cyt a3 and the Cu are involved with the reduction of O2 to H20. Cytochromes a, >, and c are in approximately equal amounts in mitochondria (the ratios vary somewhat with plant species) flavoproteins are about 4 times, ubiquinones 7 to 10 times, and pyridine nucleotides 10 to 30 times more abundant than are individual cytochromes. Likewise, in chloro-plasts the quinones and the pyridine nucleotides are much more abundant than are the cytochromes (see Table 5-3). 

The means by which NAD affects the oxidation of NADH is still uncertain. The evidence for two pyridine nucleotide binding sites is not compelling. The alternative explanation that NAD functions by reversing the equilibrium between EHj and 4-electron-reduced enzyme (EH4) is shown in Eq. (9). There is some kinetic evidence for a dead end complex... 

The principal features, in addition to EHj, common to lipoamide dehydrogenase and glutathione reductase deserve emphasis the formation of complexes between the oxidized enzymes and their respective oxidized pyridine nucleotides the formation of complexes between EHj and both oxidized and reduced pyridine nucleotides the formation of charge transfer complexes between 4-electron-reduced enzymes and oxidized pyridine... 

Complex formation between EH2 and pyridine nucleotide (NAD+) was first noted in lipoamide dehydrogenase [S7, 118). It was clear that the spectra of EH were different when the reductant was NADH or when it was dihydrolipoamide and in light of the NAD+ requirement in the oxidation of NADH, a complex of EHj with NAD+ was hypothesized. Thus, when reductant-dependent differences were observed in the spectra... 

Protection of the reductase by NADH from inhibition by thiol group reagents suggested that the enzyme formed a stable complex with pyridine nucleotide (353). Such a complex was readily demonstrated by difference spectroscopy. When the enzyme was reduced by NADH or AcPyADH a prominent positive band was observed at 317 nm (Fig. 16) this band was very small (and blue-shifted) when NADPH, a very poor substrate, was the reductant. Furthermore, addition of NAD+ following NADPH resulted in a difference spectrum identical with that produced by NADH. The dashed line in Fig. 15 represents the absorption resulting from NAD" binding. Thus, this band was attributed to a reduced enzyme-NAD complex (350). 

Structures of the native oxidized PDR from P. cepacia at 2.0 resolution and of PDR in complex with reduced NADH at 2.7 resolution have been determined (Correll et al., 1992). The enzyme folds into three domains, consisting of residues 1 to 102, 112 to 226 and 236 to 321, which bind FMN, pyridine nucleotide, and the 2Fe-2S center, respectively (Figure 11). 

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