Fig. 1.

ATP-dependent heme-efflux functions of HrtBA. (A) Growth rescue of heme-sensitive E. coli with HrtBA. Photographs of the culture test tubes after a 24-h incubation. JW0451, parental strain. (B) ATPase activity of HrtBA reconstituted in nanodiscs. Heme and PPIX dissolved in dimethyl sulfoxide (DMSO) were added at a final concentration of 10 μM in the presence of 2 mM ATP. n = 3. (C) Heme-dependent ATPase activity of HrtBA in the presence of 2 mM ATP. n = 3. (D) Kinetics of ATPase activity in the presence (square) or absence (circle) of 5 μM heme. n = 3. (E) UV-visible spectra of heme. Two micromolar of heme in buffer (red), empty nanodiscs (turquoise), and HrtBA-nanodiscs (blue). The nanodisc solutions were used at 4 μM for full binding to the added heme. (F) Schematic of heme transfer assay using a heme-binding protein. Heme-loaded HrtBA nanodisc was incubated with HasA in the presence or absence of ATP and subjected to gel filtration column chromatography. In total, 200 pmol of the proteins with 100 pmol heme was analyzed. (G) Heme transfer in the presence (+) or absence (w/o) of ATP. (H) Heme transfer in the presence (+) or absence (w/o) of AMPPNP.