ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membrane. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO–NO, Mn2+–NO, and Mn2+–Mn2+ pulsed electron-electron double resonance spectroscopy of TmrAB in a time resolved manner. This type IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+–ATP and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.
