Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides

The effects of six thiol-specific cross-linker dimaleimides, in which the distance of the two maleimide groups ranged from 7.7 to 16.8 Angstrom, on bovine heart mitochondria were studied at pH 7.2 and 0 degrees C, None of the dimaleimides affected mitochondrial proteins, but they caused significantly specific intermolecular cross-linking of the 30 kDa ADP/ATP carrier in submitochondrial particles. All the crosslinks were found to be formed specifically between two Cys(56) residues in the first loop facing the matrix, as we observed previously in intermolecular disulfide bridge formation catalyzed by copper o-phenanthroline [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol, Chem. 270, 29548-29554]. The dimerization was dependent on the cross-linking span of the dimaleimides, being maximum with the dimaleimide having a span of about 12 Angstrom. Cross-linking took place in the m-state carrier, but not in the c-state carrier, and inhibited ADP transport via the ADP/ATP carrier. We suggest that a pair of first loops with Cys(56) residues in the dimer form of the m-state carrier fluctuates widely with a most probable distance between them of about 12 Angstrom, and that this fluctuation modulates the transport activity of the ADP/ATP carrier.