Enzyme Catalysis and Regulation
Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding*

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Heme oxygenase (HO) catalyzes the O2- 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 Cys265 of HRM1 and Cys282 of HRM2, binds heme tightly. Reduction of the disulfide bond increases the Kd for ferric heme from 0.03 to 0.3 μm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the Kd for heme, they do not alter the kcat 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 Ca2+-activated potassium channel.

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This work was supported by a seed grant from National Institutes of Health Center Grant 1P20RR17675 to the University of Nebraska, and the mass spectrometry and calorimetry instruments were purchased from the same Center grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–5 and Tables S1–3.