ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a diverse array of substrates across cell membranes. We present here the dynamics of complex formation of three structurally characterized ABC transporters—the BtuCD vitamin B_(12) importer and MetNI d/l-methionine importer from Escherichia coli and the Hi1470/1 metal-chelate importer from Haemophilus influenzae—in complex with their cognate binding proteins. Similarly to other ABC importers, MetNI interacts with its binding protein with low affinity (K_d ~10^(−4) M). In contrast, BtuCD–BtuF and Hi1470/1–Hi1472 form stable, high-affinity complexes (K_d ~10^(−13) and 10^(−9) M, respectively). In BtuCD–BtuF, vitamin B_(12) accelerates the complex dissociation rate ~10^7-fold, with ATP having an additional destabilizing effect. The findings presented here highlight substantial mechanistic differences between BtuCD–BtuF, and likely Hi1470/1–Hi1472, and the better-characterized maltose and related ABC transport systems, indicating that there is considerable mechanistic diversity within this large protein super-family
