Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure

Abstract

Staphylococcus aureus IsdG catalyzes the final step of staphylococcal iron acquisition from host hemoglobin, whereby host-derived heme is converted to iron and organic products. The Asn7 distal pocket residue is known to be critical for enzyme activity, but the influence of this residue on the substrate electronic structure was unknown prior to this work. Here, an optical spectroscopic and density functional theory characterization of azide- and cyanide-inhibited wild type and N7A IsdG is presented. Magnetic circular dichroism data demonstrate that Asn7 perturbs the electronic structure of azide-inhibited, but not cyanide-inhibited, IsdG. As the iron-ligating α-atom of azide, but not cyanide, can act as a hydrogen bond acceptor, these data indicate that the terminal amide of Asn7 is a hydrogen bond donor to the α-atom of a distal ligand to heme in IsdG. Circular dichroism characterization of azide- and cyanide-inhibited forms of WT and N7A IsdG strongly suggests that the Asn7···N3 hydrogen bond influences the orientation of a distal azide ligand with respect to the heme substrate. Specifically, density functional theory calculations suggest that Asn7···N3 hydrogen bond donation causes the azide ligand to rotate about an axis perpendicular to the porphyrin plane and weakens the π-donor strength of the azide ligand. This lowers the energies of the Fe 3d xz and 3d yz orbitals, mixes Fe 3d xy and porphyrin a 2u character into the singly-occupied molecular orbital, and results in spin delocalization onto the heme meso carbons. These discoveries have important implications for the mechanism of heme oxygenation catalyzed by IsdG.

Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor

“…Later, it was established that cyanide and azide are uncompetitive inhibitors of IsdG that bind to the iron of IsdG–heme, block access for molecular oxygen, and prevent enzymatic turnover. 15 Cyanide and azide are poor drugs due to their ability to inhibit canonical heme oxygenases, 41, 42 but in principle it should be possible to design a selective, uncompetitive inhibitor for the heme to meso-hydroxyheme monooxygenation reaction or the meso-hydroxyheme to staphylobilin dioxygenation reaction (Fig. 2).…”

“…15, 24, 25 IsdG and IsdI were expressed as previously described for IsdG, 5 with one change. Expression was induced at OD 600 = 0.8 with 1.0 mM isopropyl β-D-1-thiogalactopyranoside, and cell growth continued for four hours at 37 °C.…”

“…IsdG was purified as previously described, 15 with minor changes noted below. Following cleavage of IsdG by S219V TEV protease, 50 mM imidazole was added and the solution was loaded onto a HisPur Ni-NTA column (Pierce).…”

“…15 IsdG–heme and IsdI–heme in 50 mM Tris pH 7.4, 150 mM NaCl were prepared, and their room temperature UV/Vis absorption spectra were acquired from 700 to 325 nm at a scan rate of 600 nm/min with a 1.0 nm data interval and 0.1 s integration time on a Cary 100 Bio UV-Vis Spectrophotometer. The extinction coefficients for IsdG–heme and IsdI–heme at 411 nm were found to be 98.5 mM -1 cm -1 and 96.6 mM -1 cm -1 , respectively, as determined by the pyridine hemochrome method.…”

“…1), 12-14 whereas it has been proposed that non-canonical HOs proceed through a bridged Fe–O–O–C transition state. 15, 16 Another target for selective inhibition of IsdG and IsdI in the presence of human HOs is the meso-hydroxyheme intermediate. In canonical HOs, meso-hydroxyheme is converted to verdoheme and biliverdin, 17 whereas IsdG and IsdI convert this intermediate to formyl-oxo-bilin and staphylobilin (Fig.…”