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.
Highlights
The C127A/⌬265–316 variant was generated in Heme oxygenase (HO)-2⌬265–316, whereas the other Cys variants were generated in HO-2⌬289 –316
C265A/C282A, C265A, HO-2⌬265–316, and HO-1 and HO-2 differ in their tissue distributions and expression patterns
HO-1 is inducibly expressed in most mammalian tissues, whereas HO-2 is constitutively expressed in a narrow range of tissues, with high levels in the brain and testes [11]
Summary
Overexpression, and Purification of Human HO-2— The human full-length HO-2 cDNA in a pGEX-4T-2 vector (Amersham Biosciences), which was graciously contributed by Dr Mahin D. After incubation of the 1-ml reaction mixture containing HO-2, 100 mM Tris-HCl, pH 8.0, and 100 M DTNB at room temperature for 15 min, the absorbance at 412 nm was recorded, and the free thiol concentrations were calculated by reference to a standard curve generated using dithiothreitol (DTT). After washing the pellet three times with the same solution, the protein was reduced by reacting with 10 mM DTT for 30 min at 37 °C, and the second alkylation step was performed by the addition of 2 l of 9.5 M 4-vinylpyridine and incubation for 20 min at room temperature. To determine the binding affinity of Fe3ϩ heme, freshly prepared hemin (from a stock solution of 500 M hemin in 5% Me2SO) was added in 1–2-l aliquots to the sample cuvette containing 8 M HO-2 in 1 ml of 100 mM potassium phosphate buffer, pH 7.4, and to the reference cuvette containing the same buffer. Thermogram analysis was performed using Origin Version 7.0 software supplied with the instrument
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
