Sulfitolysis and thioredoxin-dependent reduction reveal the presence of a structural disulfide bridge in spinach chloroplast fructose-1,6-bisphosphatase.

A significant difference between cytosolic and chloroplastic fructose-1,6-bisphosphatase (FbPase) is an extra peptide in the middle of chloroplast FbPase which contains three additional cysteine residues. Sit-directed mutagenesis experiments have shown that at least two of these cysteine residues are involved in forming the regulatory disulfide bridge [Jacquot, J.-P. et al., FEBS Lett. 401 (1997) 143-147] which is the presupposition for the thioredoxin-dependent control of chloroplast FbPase activity. Here we report that each subunit of the FbPase contains an additional structural disulfide bridge which has been observed by combined application of thioredoxins and sulfitolysis. Observation of the structural disulfide bridges by sulfitolysis was only possible when the FbPase was already specifically reduced by the homologous thioredoxin species TRm. and TRf from spinach chloroplasts. Interestingly, the accessibility of the structural disulfide bridge for sulfite ions depends on the thioredoxin species engaged in the thioredoxin/FbPase complex.