Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P-1B4-ATPase, in cytoplasmic Fe2+ efflux has been proposed. We report here the distinct roles of mycobacterial P-1B4-ATPases in the homeostasis of Co2+ and Fe2+. Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, an M. tuberculosis ctpJ mutant is more sensitive to Co2+ than Fe2+, whereas mutation of the homologous M. tuberculosis ctpD leads to Fe2+ sensitivity but no alterations in Co2+ homeostasis. In vitro, the three enzymes are activated by both Fe2+ and Co2+ and bind 1 eq of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe2+ and twice the Fe2+-stimulated activity than the CtpJs. These parameters are paralleled by a lower activation and affinity for Co2+. Analysis of Fe2+ and Co2+ binding to CtpD by x-ray absorption spectroscopy shows that both ions are five-to six-coordinate, constrained within oxygen/nitrogen environments with similar geometries. Mutagenesis studies suggest the involvement of invariant Ser, His, and Glu residues in metal coordination. Interestingly, replacement of the conserved Cys at the metal binding pocket leads to a large reduction in Fe2+ but not Co2+ binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe2+ levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe2+ transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress.