Abstract
Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.
Highlights
Heme oxygenase (HO)-2 appears to interlink redox and heme homeostasis; the midpoint reduction potential of the thiol/disulfide redox switch is poised near the ambient intracellular redox potential, and the Kd for heme (0.03 M) is near the free cellular heme pool (0.03–1 M) [34]
We found that HO-2 expression levels are unaffected by alteration in the cellular redox state; the thiol/disulfide switch does exhibit a robust response to redox conditions
When cells are exposed to oxidative conditions, the Cys residues in the C-terminal heme regulatory motifs (HRMs) in HO-2 are in the disulfide state, which favors heme binding; under reducing conditions, the lower affinity dithiol state predominates
Summary
Determination of the Midpoint Reduction Potential of the Thiol/Disulfide Redox Couple in HO-2—Two truncated forms of HO-2, HO-2t, which contains all three HRMs, and C127A, which contains HRM1 and HRM2 but lacks HRM3, were used in these experiments because the full-length purified protein is RTreduced HO-2͒ E ϭ Eo Ϫ zF Inoxidized HO-2͒ Intrinsic Fluorescence Quenching and CD Analysis—Tryptophan fluorescence is not detectable in as-isolated HO-2; we engineered a Phe to Trp substitution near the C-terminal HRMs. This F253W variant exhibits intrinsic tryptophan fluorescence that increases upon reduction of the disulfide bond, providing a straightforward and direct measure of the redox state of the thiol/disulfide couple.
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