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

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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.

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Abbreviations

Abs:

Electronic absorption

CD:

Circular dichroism

DFT:

Density functional theory

Fe(III)TMP(4-CNPy)2 :

Bis(4-cyanopyridine)ferrictetramesitylporphyrinate

Isd:

Iron-regulated surface determinant

IsdG–heme:

Heme-bound IsdG

IsdG–heme–CN:

Cyanide-inhibited IsdG

IsdG–heme–N3 :

Azide-inhibited IsdG

IsdG–heme–OOH:

Hydroperoxo-ligated, heme-bound IsdG

IsdI–heme–OH:

Hydroxide-ligated, heme-bound IsdI

KPi:

Potassium phosphate

MCD:

Magnetic circular dichroism

NaPi:

Sodium phosphate

PMSF:

Phenylmethanesulfonyl fluoride

TEV:

Tobacco etch virus

VTVH:

Variable-temperature, variable-field

WT:

Wild-type

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Acknowledgments

M.D.L. thanks the University of Vermont for financial support and D.S.P. thanks ACS Project SEED for summer support. We also thank Eric Skaar (Vanderbilt University) and Celia Goulding (University of California-Irvine) for the gift of the pET15b vector encoding WT His6-tagged S. aureus IsdG, David Waugh (National Cancer Institute) for providing the BL21(DE3)-RIL cell line for over-expression of S219V TEV protease, Prof. Mark Riley (University of Queensland) for providing a free copy of the VTVH MCD simulation program, and Jill Chipman (Hamilton College) for initial CD spectroscopic characterization of IsdG–heme–N3. The authors acknowledge the Vermont Advanced Computing Core which is supported by NASA (NNX 06AC88G), at the University of Vermont for providing High Performance Computing resources that have contributed to the research results reported within this paper.

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Authors and Affiliations

  1. Department of Chemistry, University of Vermont, 82 University Place, Burlington, VT, 05405, USA

    Cheryl L. Lockhart, Matthew A. Conger, Dylanger S. Pittman & Matthew D. Liptak

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  1. Cheryl L. Lockhart
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  2. Matthew A. Conger
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Correspondence to Matthew D. Liptak.

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Lockhart, C.L., Conger, M.A., Pittman, D.S. et al. Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure. J Biol Inorg Chem 20, 757–770 (2015). https://doi.org/10.1007/s00775-015-1263-5

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