FIGURE 3.

The IsdH^N3 NEAT domain binds heme. A, UV spectrum of purified IsdH^N3 that show characteristics of a heme-binding protein. These include a Soret band at 402 nm along with additional Q-band peaks at 511, 548, and 625 nm. Abs, absorbance. B, quantitative measurement of protoporphyrin binding using a fluorescence assay. The panel shows a plot of ZnPPIX fluorescence emission at 585 nm as a function of IsdH^N3 added. The solid line shows a best fit of this data which yields a K_D of ZnPPIX binding to IsdH^N3 of 2.8 ± 0.1 μm. C, an ELISA assay showing that the IsdH^N1 NEAT domain binds MetHb, whereas the IsdH^N3 does not. IsdH^N3 binding to a range of other proteins was also tested, but no interaction could be detected.