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The mitochondrial flavoenzyme L-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340, located in the cap domain. Electrospray ionization mass spectrometry revealed that the partial reversible oxidative modification of Cys-340 involves the sequential formation of sulfenic, sulfinic, and sulfonic acid states. S-Glutathionylation of the sulfenic acid switches off GALDH activity and protects the enzyme against oxidative damage in vitro. C340A and C340S GALDH variants are insensitive toward thiol oxidation, but exhibit a poor affinity for L-galactono-1,4-lactone. Cys-340 is buried beneath the protein surface and its estimated pK^sub a^ of 6.5 suggests the involvement of the thiolate anion in substrate recognition. The indispensability of a redox-sensitive thiol provides a rationale why GALDH was designed as a dehydrogenase and not, like related aldonolactone oxidoreductases, as an oxidase.
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L-Ascorbate (vitamin C) is the most consumed vitamin on earth. It is a multifunctional antioxidant that is particularly abundant in plants where it can reach millimolar concentrations, representing over 10% of the soluble carbohydrate content. L-Ascorbate is a cofactor for a number of enzymes and it is a major constituent of the intracellular redox buffer. Its main function in plants is to scavenge reducing equivalents originating from respiration and photosynthetic activity, protecting proteins, unsaturated fatty acids, and DNA from irreversible oxidative damage (Smirnoff and Wheeler, 2000).
The terminal step of L-ascorbate biosynthesis in plants is catalyzed by the mitochondrial flavoenzyme L-galactono-g-lactone dehydrogenase (GALDH; L-galactono-1,4-lactone:ferricytochrome c oxidoreductase; EC 1.3.2.3). GALDH mediates the two-electron oxidation of L-galactono-1,4-lactone into L-ascorbic acid with the concomitant reduction of cytochrome c (Scheme 1):
Besides from producing L-ascorbate, the exploitation of the electron transport chain by GALDH is important for the proper functioning of plant mitochondria (Alhagdow et al., 2007). Furthermore, it has been reported that GALDH is required for the correct assembly of respiratory complex I (Pineau et al., 2008).
GALDH and other aldonolactone oxidoreductases are two-domain proteins with a conserved vanillylalcohol oxidase (VAO)-type FAD domain (Fraaije et al., 1998; Leferink et al., 2008a). Most aldonolactone oxidoreductases are hydrogen peroxide-producing oxidases containing covalently bound FAD, while GALDH reacts poorly with molecular oxygen and contains...