Characterization of IsdH (NEAT domain 3) and IsdB (NEAT domain 2) in Staphylococcus aureus by magnetic circular dichroism spectroscopy and electrospray ionization mass spectrometry

Abstract

Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.