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An investigation of hemopexin redox properties by spectroelectrochemistry: biological relevance for heme uptake

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Abstract

Hemopexin (HPX) has two principal roles: it sequesters free heme in vivo for the purpose of preventing the toxic effects of this moiety, which is largely due to heme’s ability to catalyze free radical formation, and it transports heme intracellularly thus limiting its availability as an iron source for pathogens. Spectroelectrochemistry was used to determine the redox potential for heme and meso-heme (mH) when bound by HPX. At pH 7.2, the heme-HPX assembly exhibits E 1/2 values in the range 45–90 mV and the mH-HPX assembly in the range 5–55 mV, depending on environmental electrolyte identity. The E 1/2 value exhibits a 100 mV positive shift with a change in pH from 7.2 to 5.5 for mH-HPX, suggesting a single proton dependent equilibrium. The E 1/2 values for heme-HPX are more positive in the presence of NaCl than KCl indicating that Na+, as well as low pH (5.5) stabilizes ferro-heme-HPX. Furthermore, comparing KCl with K2HPO4, the chloride salt containing system has a lower potential, indicating that heme-HPX is easier to oxidize. These physical properties related to ferri-/ferro-heme reduction are both structurally and biologically relevant for heme release from HPX for transport and regulation of heme oxygenase expression. Consistent with this, when the acidification of endosomes is prevented by bafilomycin then heme oxygenase-1 induction by heme-HPX no longer occurs.

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Abbreviations

DMEM:

Dulbecco’s Minimal Essential Medium

HEPES:

4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

H:

Heme, protoporphyrin-IX

HO-1:

Heme oxygenase-1

HPX:

Hemopexin

mH:

Meso-heme

NHE:

Normal hydrogen electrode

OTTLE:

Optically transparent thin-layer electrode

SDS:

Sodium dodecyl sulfate

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Acknowledgments

A.L.C. thanks the National Science Foundation (Grants CHE 0079066 and CHE 0418006) for financial support. M.M.F. is a Duke University Toxicology Program Fellow supported by NIEHS grant T32-ES-07031-21A1. A.S. thanks NIH (Grant DK64363), the American Heart Association and the University of Missouri Research Board for financial support. The authors acknowledge the help of Ms. Rachel Lovelace and Dr. Max Paoli (University of Newcastle, UK) for the generation of the heme-hemopexin structure with PyMol.

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Authors and Affiliations

  1. Department of Chemistry, Duke University, Box 90346, Durham, NC, 27708-0346, USA

    Meghan M. Flaherty & Alvin L. Crumbliss

  2. Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri – K.C., Kansas City, MO, 64110-2499, USA

    Kimberley R. Rish & Ann Smith

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  1. Meghan M. Flaherty
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  2. Kimberley R. Rish
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  3. Ann Smith
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  4. Alvin L. Crumbliss
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Correspondence to Alvin L. Crumbliss.

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Flaherty, M.M., Rish, K.R., Smith, A. et al. An investigation of hemopexin redox properties by spectroelectrochemistry: biological relevance for heme uptake. Biometals 21, 239–248 (2008). https://doi.org/10.1007/s10534-007-9112-9

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