Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus
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, Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus, Journal of Biological Chemistry, 293, (18) pp. 6942-6957. ISSN 0021-9258 (2018) [Refereed Article]
Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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
(IsdHN2N3) triggers hemin release by distorting Hb’s F-helix.
Here, we report that IsdHN2N3 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 IsdHN2N3-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.