ROLE OF CYS RESIDUES AS A THIOL/DISULFIDE SWITCH IN HEME OXYGENASE 2 PROTEIN

University Of California, San Francisco, San Francisco CA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heme oxygenases (HO) are enzymes involved in degradation of Fe(III) protoporphyrin IX (heme) to biliverdin IX, Fe(II) and carbon monoxide (CO) in the presence of O2 and NADPH cytochrome P450 reductase. The HOs are the only enzymes that can degrade heme indicating their essential role in maintaining heme and iron homeostasis. In addition, the three products generated from HO catalysis have important biological functions. The iron is recycled since only ~3% of the amount of iron required daily is obtained by the diet. CO serves as a signaling molecule exhibiting anti-apoptotic, anti-inflammatory, and anti-proliferation properties. Whereas, biliverdin is reduced by biliverdin reductase (in mammals) to the potent antioxidant bilirubin, which is subsequently conjugated with glucoronic acid and later secreted. The HO2 protein consists of three Cys residues which are proposed to regulate the binding affinity of HO2 for heme. The presence or absence of the disulfide bond formed between residues Cys265 and Cys282 resulted in an ~10-fold difference in the HO2 affinity for heme suggesting an important role of Cys in regulating the cellular levels of the free heme, CO, Fe, and biliverdin. There is no structural information of the region comprising C265 and C282 residues since they are missing in the HO2 crystal structure and furthermore, no NMR structural data has been reported up to date. We are using (1H, 13C) 2 dimensional NMR spectroscopy to monitor the redox state of Cys residues and protein conformational changes upon heme ligand binding. The HO2 proteins used for NMR measurements are L-[3-13C]-cysteine labeled and the incorporation efficiency of isotopically labeled Cys will be determined by Mass Spectrometry analysis. Furthermore, we will determine the redox state of the Cys residues by absorbance spectroscopy and Mass Spectrometry analysis.