CONVERSION OF THE FHUA TRANSPORT PROTEIN INTO A DIFFUSION CHANNEL THROUGH THE OUTER-MEMBRANE OF ESCHERICHIA-COLI

The FhuA receptor protein is involved in energy-coupled transport of F e3+ via ferrichrome through the outer membrane of Escherichia coli. Si nce no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD prot eins, which are anchored to the cytoplasmic membrane. By deleting 34 a mino acid residues of a putative cell surface exposed loop, FhuA was c onverted from a ligand specific transport protein into a TonB independ ent and nonspecific diffusion channel. The FhuA deletion derivative Fh uA DELTA322-355 formed stable channels in black lipid membranes, in co ntrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at leas t three times larger than that of the general diffusion porins of E.co li outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-a ctive conformation of FhuA is mediated by a TonB-induced conformationa l change in response to the energized cytoplasmic membrane. The ferric hrome transport rate into cells expressing FhuA DELTA322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 muM) , in contrast to FhuA wild-type cells, which displayed saturation at 5 muM. This implies that in wild-type cells ferrichrome transport throu gh the outer membrane is the rate-limiting step and that TonB, ExbB an d ExbD are only required for outer membrane transport.