Lipoic acid disulfide, reduction

Lipoic acid exists as a mixture of two structures a closed-ring disulfide form and an open-chain reduced form (Figure 18.33). Oxidation-reduction cycles interconvert these two species. As is the case for biotin, lipoic acid does not often occur free in nature, but rather is covalently attached in amide linkage with lysine residues on enzymes. The enzyme that catalyzes the formation of the lipoamide nk.2Lg c requires ATP and produces lipoamide-enzyme conjugates, AMP, and pyrophosphate as products of the reaction. 

The special properties of lipoic acid arise from the ring strain experienced by oxidized lipoic acid. The closed ring form is approximately 20 kj higher in energy than the open-chain form, and this results in a strong negative reduction potential of about —0.30 V. The oxidized form readily oxidizes cyanides to isothiocyanates and sulfhydryl groups to mixed disulfides. 

In lipoic acid (6), an intramolecular disulfide bond functions as a redox-active structure. As a result of reduction, it is converted into the corresponding dithiol. As a prosthetic group, lipoic acid is usually covalently bound to a lysine residue (R) of the enzyme, and it is then referred to as lipoamide. Lipoamide is mainly involved in oxidative decarboxylation of 2-0X0 acids (see p. 134). The peptide coenzyme glutathione is a similar disulfide/ dithiol system (not shown see p. 284). 

The reaction catalyzed by the first of these is illustrated in Table 15-2 (reaction type F). The other two enzymes usually promote the reverse type of reaction, the reduction of a disulfide to two SH groups by NADPH (Eq. 15-22). Glutathione reductase splits its substrate into two halves while reduction of the small 12-kDa protein thioredoxin (Box 15-C) simply opens a loop in its peptide chain. The reduction of lipoic acid opens the small disulfide-containing 5-membered ring in that molecule. Each of these flavoproteins also contains within its structure a reducible disulfide group that participates in catalysis. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

Howie, J.K., Houts, J.J., and Sawyer, D.T. 1977. Oxidation-reduction chemistry of DL-a-lipoic acid, propanedithiol, and trimethyllene disulfide in aprotic and in aqueous media. Journal of the American Chemical Society 99, 6323-6326. 

Partial protection from scurvy in guinea pigs provided by a-lipoic acid (R22) is probably due to the preservations of small residual amounts of ascorbic acid in the diet. Ascorbic acid has similar effects on riboflavin-(T4) and thiamine-deficient (T3) rats. The temporary improvements are the result of the reduction of thiamine disulfide to nutritionally active forms (B3). 

Reduction of Fe(IV) = 0 Lb by disulfides does not occur to any significant extent, except in the case of oxidized lipoic acid, which brings about slow reduction of Fe(IV) = 0 Lb to Fe(III) Lb (135). No radical species were detected by EPR spectroscopy during the reaction, though such species are very likely generated by one-electron oxidation of the disulfide. No reaction between Fe(IV) = 0 Lb and hydrogen sulfide has been observed, unlike the situation with myoglobin (137). 

Lipoic acid is required by many microorganisms for proper growth. As a disulfide, it functions in the living system by catalyzing certain oxidation reactions and is reduced in the process. Write the structure of the reduction product. 

A soln. of lipoic acid in acetaldehyde irradiated 1-2 hrs. under Ng below 5° with X > 330 nm liq.-filtered light of a 100 w. high-pressure Hg-lamp product. Y 78%. F. e., also reduction of open-diain disulfides, s. M. Takagi, S. Goto, and T. Matsuda, Chem. Commun. 1976, 92. 

If the theory of the mode of action of lipoic acid is correct, the reduced dithiol form of a-lipoic acid is equally as important as the disulfide ring form and, in fact, the two are believed to form a reversible oxidation-reduction couple, as is shown in equation 10. 

The polymer-bound lipoic acid can be reduced by using NaBH4 (see Scheme 15-5). The reduced polymers are stable between pH 2-10 and can be reused after reduction. The resulting reduced polymer bisthiol is a potential reducing agent for disulfides. The degree of lipoyl substitution in the polymer can be found by reduction of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) (Ellman, 1959). The thiol content of the polymer can also be determined by reaction of the polymer with iodo[ C]acetic acid and by measurement of the uptake of radioactivity by liquid scintillation counting. 

In view of the fact that lipoic acid is a cyclic disulfide, it is tempting to speculate on its function as a 2-carbon carrier in pyruvate oxidation through a thioester in a fashion similar to the CoA fimction in this reaction. Similarly the disulfide nature of this factor invites speculation on its possible role as an oxidation-reduction coenzyme by going through a sulfhydryl form. It is a curious fact that three of the factors involved in pyruvate oxidation, thiamine pyrophosphate, CoA, and POF, all contain sulfur. In the first two of these factors, the sulfur appears to play an important role in their mechanism of action. By analogy one would suppose that the disulfide grouping of POF will be prominent in its mechanism of action. 

It is easy to recognise that the reductive cleavage, forming freely diffusing thiyl radicals, is not possible when the disulfide bridge is held together by a linking backbone as, for example, in lipoic acid and several cyclic disulfides. Reduction of these compounds have been examined extensively and lifetimes of over lOOps have been measured for the 2c/3e bonded radical anion, even in the absence of free thiolate [14, 28-31]. Here, as depicted schematically in equation (15) the thiyl and thiolate components are prevented from free diffusion and, therefore, the back reaction of the equilibrium will be much faster and more efficient than in the non-linked systems. 

Disulfide radical cations are reasonably good oxidants. Some representative reduction potentials, measured for the dimethyldisulfide and lipoic acid systems [162] are listed in Table 9. 

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