Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase

Oxidation-reduction midpoint potentials (E-m) have been measured for the thioredoxin-dependent, reductive activation of sorghum nicotinamide adenine dinucleotide phosphate- (NADP-) dependent malate dehydrogenase (MDH) in the wild-type enzyme and in a number of site-specific mutants, The E-m value associated with activation of the wild-type enzyme, -330 mV at pH 7.0, can be attributed to the E-m of the C365/C377 disulfide present in the C-terminal region of the enzyme. The C24/C29 disulfide, located in the N-terminal region of the enzyme and the only other disulfide present in oxidized, wild-type MDH, has a E-m value of -280 mV at pH 7.0, A third regulatory disulfide, C23/C207, that is absent in the oxidized enzyme but is thought to be formed during the activation process, has an E-m value at pH 7.0 of -310 mV. E-m vs pH profiles suggest pK(a) values for the more acidic cysteine involved in the formation of each of these disulfides of 8.5 for C24/C29; 8.1 for C23/C207; and 8.7 for C365/C377. The results of this study show that the N-terminal disulfide formed between C24 and C29 has a more positive E-m value than the two other disulfides and is thus is likely to be the "preregulatory disulfide" postulated to function in activating the enzyme.