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Abstract

Staphylococcus aureus is a leading cause of life-threateninginfections in the United States. It actively acquires the essentialnutrient iron from human hemoglobin (Hb) using the iron-regulatedsurface-determinant (Isd) system. This process is initiatedwhen the closely related bacterial IsdB and IsdH receptorsbind to Hb and extract its hemin through a conserved tri-domainunit 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(IsdHN2N3) triggers hemin release by distorting Hb’s F-helix.Here, we report that IsdHN2N3 promotes hemin release fromboth the - and -subunits. Using a receptor mutant that onlybinds to the -subunit of Hb and a stopped-flow transfer assay,we determined the energetics and micro-rate constants ofhemin 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. Wepropose that hemin removal requires the rate-limiting hydrolyticcleavage of the axial HisF8 N±–Fe3± bond, which, basedon molecular dynamics simulations, may be facilitated byreceptor-induced bond hydration. Isothermal titration calorimetryexperiments revealed that two distinct IsdHN2N3-Hbproteinprotein interfaces promote hemin release. A high-affinityreceptorHb(A-helix) interface contributed -95% ofthe total binding standard free energy, enabling much weakerreceptor interactions with Hb’s F-helix that distort its heminpocket and cause unfavorable changes in the binding enthalpy.We present a model indicating that receptor-introduced structuraldistortions and increased solvation underlie the IsdH-mediatedhemin extraction mechanism.

Item Type:	Article
Authors/Creators:	Sjodt, M and Macdonald, R and Marshall, JD and Clayton, J and Olson, JS and Phillips, M and Gell, DA and Wereszczynski, J and Clubb, RT
Keywords:	haemoglobin, staphylococcus aureus
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	Amer Soc Biochemistry Molecular Biology Inc
ISSN:	0021-9258
DOI / ID Number:	10.1074/jbc.RA117.000803
Copyright Information:	Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Related URLs:	UTAS Profile: Gell, DA
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