Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN^2N3) triggers hemin release by distorting Hb’s F-helix. Here, we report that IsdH^N2N3 promotes hemin release from both the - and -subunits. Using a receptor mutant that only binds to the -subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400- fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 N±–Fe3± bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdH^N2N3-Hb proteinprotein interfaces promote hemin release. A high-affinity receptorHb(A-helix) interface contributed -95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb’s F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.

Item Type:	Refereed Article
Keywords:	haemoglobin, Staphylococcus aureus
Research Division:	Biological Sciences
Research Group:	Biochemistry and cell biology
Research Field:	Structural biology (incl. macromolecular modelling)
Objective Division:	Expanding Knowledge
Objective Group:	Expanding knowledge
Objective Field:	Expanding knowledge in the biological sciences
UTAS Author:	Gell, DA (Dr David Gell)
ID Code:	128723
Year Published:	2018
Web of Science® Times Cited:	9
Deposited By:	Medicine
Deposited On:	2018-10-10
Last Modified:	2019-04-08
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