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Format:
Print
Author:
Lockhart, Cheryl
Dept./Program:
Chemistry
Year:
2014
Degree:
MS
Abstract:
Iron is a necessary nutrient for pathogens such as Staphylococcus aureus but free iron is not readily available during infection. The majority of iron in the human body is heme-iron, and S. aureus employs a heme uptake system to acquire heme and transport it to the bacterial cytoplasm. There, the heme oxygenase IsdG is one enzyme that degrades the porphyrin ring to release free iron. The active site residue Asn7 is essential for enzymatic turnover and here it is proposed that hydrogen bond donation from the Asn7 side-chain to a distal heme ligand perturbs the substrate electronic structure. This hydrogen bonding interaction may promote the heme degradation reaction catalyzed by IsdG.
We have employed site-directed mutagenesis, a variety of distal ligands to the heme substrate, and optical spectroscopy to investigate whether a hydrogen bond between Asn7 and a distal ligand exists that is strong enough to perturb the substrate electronic structure. The electronic absorption (Abs), circular dichrosim (CD), and magnetic CD (MCD) spectra of azide-and cyanide-inhibited wild type (WT) and N7A IsdG have been compared to identify the effects that the Asn7 side-chain has on the heme electronic structure. In this study, azide (N₃⁻) was employed to mimic the hydrogen bond accepting properties of the putative peroxo-ligated intermediate of IsdG-catalyzed heme degradation. In contrast, the metal-ligating carbon of cyanide (CN⁻) cannot hydrogen bond.
Abs spectroscopy of uninhibited WT and N7A IsdG-heme implies that hydrogen bond donation from Asn7 lowers the pKa of a distal water ligand. The sign of the Soret CD band for IsdG-heme-N₃ reverses upon introduction of the N7A mutation, suggesting that this mutation alters the orientation of the distal azide ligand relative to the porphyrin. Significant differences were observed between the MCD spectra of WT and N7A IsdG-heme-N₃, whereas the spectrum of IsdG-heme-CN is not perturbed by the N7A mutation. These data are consistent with hydrogen bond donation from Asn7 to the ironligated atom of the distal ligand with the IsdG active site altering the heme substrate electronic structure.
Additional analysis of the MCD spectroscopic data, specifically measurement of variable-temperature, variable-field saturation magnetization curves, suggests that two electronic states are populated in WT IsdG-heme-CN, whereas only one state is populated for the IsdG-heme-N₃. These data are discussed with respect to their implications for the rate and regioselectivity of the IsdG-catalyZed heme degradation.