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Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding.

ABSTRACT: Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.

SUBMITTER: Yi L

PROVIDER: S-EPMC3957417 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding.

Yi Li L Ragsdale Stephen W SW

The Journal of biological chemistry 20070531 29

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calori ...[more]

PMID: 17540772

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Project description:Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O2- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2(O)), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2(R)) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme.

Dataset Information

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding.

Publications

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding.

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