Ascorbic acid reaction with cytochrome

While XH2 could be identical with ascorbic acid itself, it is more likely to be another reduced substance, e.g. NADHj, NADPHj, a-ketoglutar-ate, etc., with ascorbic acid and a cytochrome playing a cycling redox role as shown in Figure 5.11. In some cases ascorbic acid may act as substance XHj and the reaction could be simplified to ... 

Ascorbic acid is highly reactive to all the primary water radicals, because of its carbonyl group and double bond. Reaction with ei or H reduces ascorbic acid to a ketyl radical, while reaction with OH oxidizes it to the relatively unreactive tricarbonyl radical ion [9]. Aside from a possible reaction with cytochrome-c (or ferrimyoglobin), the radical ion is most likely to undergo a complex disproportionation reaction that regenerates the ascorbic acid and produces dehydroascorbic acid, which has essentially the same vitamin activity. These reactions need to be considered, because ascorbic acid is added to foods to fortify them, to facilitate curing meats, and to enhance antioxidants. 

L-Tyrosine metabohsm and catecholamine biosynthesis occur largely in the brain, central nervous tissue, and endocrine system, which have large pools of L-ascorbic acid (128). Catecholamine, a neurotransmitter, is the precursor in the formation of dopamine, which is converted to noradrenaline and adrenaline. The precise role of ascorbic acid has not been completely understood. Ascorbic acid has important biochemical functions with various hydroxylase enzymes in steroid, dmg, andhpid metabohsm. The cytochrome P-450 oxidase catalyzes the conversion of cholesterol to bUe acids and the detoxification process of aromatic dmgs and other xenobiotics, eg, carcinogens, poUutants, and pesticides, in the body (129). The effects of L-ascorbic acid on histamine metabohsm related to scurvy and anaphylactic shock have been investigated (130). Another ceUular reaction involving ascorbic acid is the conversion of folate to tetrahydrofolate. Ascorbic acid has many biochemical functions which affect the immune system of the body (131). 

Cytochrome c is a heme containing protein which occurs in muscle at lower concentrations than does myoglobin. It was demonstrated some time ago (18) that oxidized cytochrome c reacts with gaseous nitrite oxide to produce a nltrosyl compound. Recent work (19, 20, 21) has examined the reactions of cytochrome c with nitrite and the contribution of the product formed to cured meat color in considerably more detail. The general conclusion is that even at the pH normally encountered in meat, the reaction can take place in the presence of ascorbic acid but probably does not affect meat color because of the unstable nature of the reaction product and the low concentration. 

Staudinger s interpretation of this involves the central problem of such hydroxylation reactions the generation of free radicals from O2 as the possible hydroxylating agents (K4). The hypothesis is that the reaction of TPNH with O2 in Ae mitochondrial 11-P-hydroxylase generates such free radicals, and so does the oxidation of ascorbic acid by the DPNH-oxidase-cytochrome be system (Eq. 15). The monodehydroascorbic acid formed in the latter system is already well documented. 

There are several examples reported in the literature that attempt to combine all the elements of the cytochrome P-450 system with the goal of catalyzing epoxidation reactions, all with varying degrees of success. Some of these systems have used a variety of electron sources including sodium borohydride,l7 colloidal platinum/H2, zinc powder, and ascorbic acid.20... 

Strong pH dependencies of the rates of reduction of a number of cytochromes c and the blue copper protein azurin by ascorbic acid are interpreted in terms of reaction of the ascorbate anion rather than highly reactive deprotonated forms of the proteins since it is unlikely that all the proteins would act in a similar fashion. The second-order rate constants for the ascorbate " anion increase with increasing positive charge on the protein consistent with an outer-sphere mechanism. This is confirmed by cyt c(iii) where the rate of reduction by ascorbate exceeds the limiting rate of substitution of methionine-80 by imidazole at pH 9.0 (7=0.1 M, 21 °C), 8 s" Activation parameters are similar to those of other outer-sphere reactions between proteins and small-molecule redox agents. 

As discussed previously, cytochrome c is reduced by several flavo-proteins. On enzymatic reduction or reaction with hydrosulfite, ascorbic acid, or any of several other reducing reagents, the typical spectrum of reduced cytochrome c is produced. The band at 550 m/ is usually used to assay reduced cytochrome c. The reduction causes a change in the iron from the ferric to the ferrous state. The oxidation-reduction potential of the couple, ferric3rtochrome Jerrocytochrome at neutral pH values (5-8) is -H0.256 volts. ... 

Many attempts have been made to demonstrate phosphorylation coupled to oxidation of reduced cytochrome c. Judah has presented evidence which suggests that phosphorylation occurs when cytochrome c is reduced in situ by a non-enzymatic reaction with ascorbic acid, and then subsequently oxidized enzymatically. Slater, on the other hand, has reported experiments which he interprets as meaning that phosphorylation does not occur when cytochrome c is oxidized. Slater s experiments seem to be open to some criticism on technical grounds. In any event, it is difficult to explain the P/0 ratio of 2.0 which has been observed in the oxidation of succinate unless one assumes that phosphorylation occurs coupled to oxidations above the level of cytochrome c. The potential span between succinate and cytochrome c is only about 0.3 v., which is not sufficient to generate the 24,000 cal. needed to bring about the formation of 2 moles of ADP to ATP. 

Fig. 10. Reduction of cytochrome a by /)-phenylenediamine, hydroquinone, and ascorbic acid. The sample cuvette contained 2.7 ml. of cytochrome a solution diluted with 0.1 M phosphate buffer containing 1% Emasol 4130 at pH 7.4 (4.6 X 10 M cytochrome a). The reaction was initiated by the addition of 0.3 ml. of 0.01 M solutions of each reagent. The reduction was determined by measuring the increase in optical density of the a band at 605 mj or of the y band at 444 mu in a Cary spectrophotometer at 30°C. Curves A, B, and C show the reduction of the a band by p-pbenylenediamine, hydroquinone, and ascorbic acid, respectively. Curve D shows the reduction of the y band by p-phenylenediamine using a concentration of cytochrome a which was one-third of that used for the other curves.

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