Time‐Resolved Mn²⁺−NO and NO−NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter**

Abstract

ATP‐binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. 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 the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy

“…Nitroxide probes are frequently used because they are stable radicals with spin-Hamiltonian parameters and relaxation properties that are well suited for detection with PDS techniques. − Paramagnetic metal ions like Fe 3+ , Mn 2+ , Co 2+ , Ni 3+ , Cu 2+ , and Gd 3+ can be used for PDS as well, − and some of these ions exist as naturally occurring cofactors in enzymes. Another possibility to make a biopolymer EPR-active is to substitute diamagnetic Mg 2+ with paramagnetic Mn 2+ . , …”