Dehydrogenases direct coenzyme transfer

An intriguing generalization was recently proposed by Srivastava and Bern-hard who suggested that pairs of different dehydrogenases capable of transferring coenzyme directly between their active sites via an intermediate ternary complex are of opposite stereochemistries (52) [Eq. (6)] ... 

This was the first example of a reduction of an aldehyde by an NADH analogue in a nonenzymatic system. If 1-propyl-4,4-dideuterionicotinamide is used, monodeuterated l,10-phenanthroline-2-carbinol is produced. This result demonstrates that the product is formed by direct hydrogen transfer from the reduced coenzyme analogue. It strengthens the view that coordination or proximity of the carbonyl to the zinc ion is probably important in the enzymatic catalysis. Zn(II) ion is known to be essential for the catalytic activity of horse liver alcohol dehydrogenase (280). 

Last, let us consider the possibility of a mechanism other than a hydride transfer in NAD chemistry. Indeed, G. A. Hamilton argued that if a direct hydride transfer process occurs in dehydrogenase reactions, it is unique in biology since proton transfer would be more favorable (279). However, it is not a simple task to distinguish between these two possibilities. Generally, it is simpler to say that the reduction reaction is analogous to a transfer of two electrons rather than postulating a hydride ion. More will be said on this subject in Section 7.1.3 on flavin coenzyme. 

Specific redox characteristics of a catalyst derived from CV scans are also used to confirm an enzyme s ability for bioelectrocatalysis by either direct electron transfer (DET) or mediated electron transfer (MET) to the electrode. DET and MET are two distinct mechanisms of bioelectrocatalysis. MET has the advantage of being compatible with almost all naturally occurring oxidoreductase enzymes and coenzymes, but it requires additional components (either smaU-molecule redox mediators or redox polymers) because the enzymes cannot efficiently transfer electrons to the electrode. These additional components make the system more complex and less stable [8]. The vast majority of oxidoreductase enzymes that require MET to an electrode are nicotinamide adenine dinucleotide (NAD" ) dependent. Two of the most commonly encountered NAD -dependent enzymes in BFC anodes are glucose dehydrogenase (GDH) and alcohol dehydrogenase (ADH). These enzymes have been thoroughly characterized in respect to half-cell electrochemistry and have been demonstrated for operation in BFC. More information about MET can be found in Chapter 9. 

By using dideuterioethanol, CH3CD2OH, as substrate, they demonstrated that one atom of deuterium is transferred from substrate to coenzyme, and further that this atom of deuterium is transferred from the coenzyme, under the influence of lactic dehydrogenase, to pyruvate (Fisher, et al., 1953 Loewus et al., 1953a,b Vennesland and Westheimer, 1954). The converse experiment was also carried out, that is to say, the reactions were performed with normal substrate and coenzyme, but in D20 this resulted in transfer of ordinary hydrogen from substrate to coenzyme. The question they had set out to answer was then settled the oxidation-reductions proceed by direct transfer of hydrogen between substrate and coenzyme. These results have subsequently been confirmed in numerous investigations with other enzymic reactions that require NAD+ or NADP+. 

The answer is d. (Murray, pp 123—148. Scriver, pp 2367—2424. Sack, pp 159—175. Wilson, pp 287-317.) Some monooxygenases found in liver endoplasmic reticulum require cytochrome P450. This cytochrome acts to transfer electrons between NADPH, O2, and the substrate. It can be an electron acceptor from a flavoprotein. In the mitochondrial electron transport chain, flavoproteins donate electrons to coenzyme Q, which then transfers them to other cytochromes. Flavoproteins that are oxidases often react directly with molecular oxygen to form hydrogen peroxide. Flavoproteins can be NADH dehydrogenases that oxidize NADH and transfer the electrons to coenzyme Q. The electron transfer centers of flavoproteins in the electron transport chain contain nonheme iron and sulfur. 

Fig. 20.6. Succinate dehydrogenase contains covalently bound FAD. As a consequence, succinate dehydrogenase and similar flavopro-teins reside in the inner mitochondrial membrane where they can directly transfer elechons into the electron transport chain. The elechons are hansferred from the covalently bound FAD to an Fe-S complex on the enzyme, and then to coenzyme Q in the electron hansport chain (see Chapter 21). Thus, FAD does not have to dissociate from the enzyme to transfer its electrons. All the other enzymes of the TCA cycle are found in the mitochondrial mahix.

Further studies [67] of enzyme/polypyrrole systems have focused on modification of the enzyme. It was found that the redox dye, Meldola blue, forms a strong complex with alcohol dehydrogenase. It is also known that this dye makes the electrochemical regeneration of the coenzyme NADH possible [68,69]. By electropolymerizing pyrrole, Meldola blue, alcohol dehydrogenase and NAD a membrane was prepared that oxidized ethanol apparently by a direct transfer of electrons to the electrode. 

Succinate dehydrogenase (EC 1.3.99.1) an oligomeric flavoenzyme [M, (bovine heart) 100,000] of the TCA-cycle, which catalyses the oxidation of succinate to fumarate. S. d. consists of two nonidentical, iron-containing subunits (Af, 70,000 and 30,000). Coenzyme of S. d. is FAD, which receives hydrogen directly (without the participation of NAD or NADP) and transfers it to ubiquinone or cytochrome b of the respiratory chain, or (in vitro) to redox dyes, e.g. methylene blue. S.d. occurs only in mitochondria (and prokaryotic cell membranes), where it is firmly bound in the membrane in association with the respiratory chain. It has a regulatory role in the TCA cycle, and can be isolated from the succinoxidase complex. 

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