Iron(III)hydroxamate transport of Escherichia coli K12: single amino acid replacements at potential ATP-binding sites inactivate the FhuC protein

Abstract

The mechanism of iron(III)hydroxamate transport appears to be of the periplasmic binding protein dependent transport (PBT) kind which is energized by ATP hydrolysis. The FhuC protein contains two domains typical of ATP-binding proteins. Lysine in domain I was replaced by glutamine and glutamate, and aspartate in domain II by asparagine and glutamate, resulting in FhuC derivatives which no longer transported ferrichrome and albomycin. FhuC inactivation by the aspartate-glutamate substitution is especially noteworthy since the negative charge thought to be involved in Mg2(+)-ATP binding remains the same and the two amino acid side chains differ in only a CH2 group. It is concluded that the two domains that represent consensus sequences among all peripheral cytoplasmic membrane proteins of PBT systems are involved in substrate transport.