Abstract
The mechanism by which transmembrane reductases use a single pair of cysteine residues to relay electrons between protein substrates across biological membranes is a long-standing mystery in thiol-redox biochemistry. Here we show the NMR structure of a reduced-state mimic of archaeal CcdA, a protein that transfers electrons across the inner membrane, by using a redox-active NMR sample. The two cysteine positions in CcdA are separated by 20 Å. Whereas one is accessible to the cytoplasm, the other resides in the protein core, thus implying that conformational exchange is required for periplasmic accessibility. In vivo mixed disulfide-trapping experiments validated the functional positioning of the cysteines, and in vitro accessibility results confirmed conformational exchange. Our NMR and functional data together show the existence of multiple conformational states and suggest a four-state model for relaying electrons from cytosolic to periplasmic redox substrates.