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Heme regulatory motifs in heme oxygenase-2 form a thiol/disulfide redox switch that responds to the cellular redox state.

Yi L, Jenkins PM, Leichert LI, Jakob U, Martens JR, Ragsdale SW - J. Biol. Chem. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

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.

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Major structural regions in HO-1 and HO-2. His25 in HO-1 or His45 in HO-2 is the heme-binding ligand.
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Figure 1: Major structural regions in HO-1 and HO-2. His25 in HO-1 or His45 in HO-2 is the heme-binding ligand.

Mentions: HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12, 13); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys265 in HRM1 and Cys282 in HRM24 (12, 14) (Fig. 1). It was shown recently that the HRMs in HO-2 do not bind heme per se but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15).


Heme regulatory motifs in heme oxygenase-2 form a thiol/disulfide redox switch that responds to the cellular redox state.

Yi L, Jenkins PM, Leichert LI, Jakob U, Martens JR, Ragsdale SW - J. Biol. Chem. (2009)

Major structural regions in HO-1 and HO-2. His25 in HO-1 or His45 in HO-2 is the heme-binding ligand.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742820&req=5

Figure 1: Major structural regions in HO-1 and HO-2. His25 in HO-1 or His45 in HO-2 is the heme-binding ligand.
Mentions: HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12, 13); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys265 in HRM1 and Cys282 in HRM24 (12, 14) (Fig. 1). It was shown recently that the HRMs in HO-2 do not bind heme per se but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

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.

Show MeSH