Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.