Structure/function relationships of the human mitochondrial ornithine/citrulline carrier by Cys site-directed mutagenesis. Relevance to mercury toxicity.

ABSTRACT

The effect of SH reagents on the human mitochondrial ornithine/citrulline carrier (hORC) was studied. Site-directed Cys mutants were employed to gain information on structure/function relationships. The substitutions of each Cys by Ala did not alter the hORC activity measured as [3H]ornithine/ornithine antiport in proteoliposomes. N­ethylmaleimide inhibited the transport of WT with IC50 of 149 µM. C51A, C50A and C132A showed a much higher IC50. MTSEA and MTSET also inhibited the WT with IC50 of 0.40 µM and 1.60 µM, respectively. C51A and C132A showed much higher IC50 values for both reagents. The triple mutant C50/51/132A showed an IC50 for the three reagents that was higher than that of the single mutants. The data strongly suggests that C132, C50 and C51 are involved in inhibition of hORC. Inhibition of WT and mutants by CuPhenanthroline, an S-S forming reagent, suggested that C132 may form disulfides with C50 or C51, impairing the transporter function. The structure/function relationships information deriving from the inhibition studies, were corroborated by the homology structural model of the transporter. The effect of HgCl2 and methyl mercury was also tested on hORC in the light of their capacity to bind thiol residues. Both reagents potently inhibit the transporter.