Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2

Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI 53706, USA.
European Journal of Biochemistry (Impact Factor: 2.54). 09/2010; 15(7):1117-27. DOI: 10.1007/s00775-010-0672-8
Source: PubMed


Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme-HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme's distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.

Download full-text


Available from: Stephen W Ragsdale,
11 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This review focuses on thiol/disulfide redox switches that regulate heme binding to proteins and modulate their activities. The importance of redox switches in metabolic regulation and the general mechanism by which redox switches modulate activity are discussed. Methods are described to characterize heme-binding sites and to assess their physiological relevance. For thiol/disulfide interconversion to regulate activity of a system, the redox process must be reversible at the ambient redox potentials found within the cell; thus, methods (and their limitations) are discussed that can address the physiological relevance of a redox switch. We review recent results that define a mechanism for how thiol/disulfide redox switches that control heme binding can regulate the activities of an enzyme, heme oxygenase-2, and an ion channel, the BK potassium channel. The redox switches on these proteins are composed of different types of Cys-containing motifs that have opposite effects on heme affinity, yet have complementary effects on hypoxia sensing. Finally, a model is proposed to describe how the redox switches on heme oxygenase-2 and the BK channel form an interconnected system that is poised to sense oxygen levels in the bloodstream and to elicit the hypoxic response when oxygen levels drop below a threshold value.
    Antioxidants & Redox Signaling 03/2011; 14(6):1039-47. DOI:10.1089/ars.2010.3436 · 7.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophages exert prominent effects in the defense of the respiratory tract from airborne pathogens. These cells are specialized to recognize, phagocytose, and destroy these infectious agents and then promote appropriate tissue repair after successful pathogen clearance. For reasons that are not presently clear, macrophages appear to be inappropriately activated during asthma responses. Evidence stems from the appearance of either classically (or M1) and alternatively activated (or M2) cells in the alveolar compartment of asthmatic lung. Macrophages localized in the interstitial area of the lung appear to be less prone to polarization toward either the M1 or M2 phenotype as these cells predominately express interleukin-10 and exhibit immunoregulatory properties. Effective treatment of clinical asthma, regardless of severity, might depend on restoring an appropriate balance between M1, M2, and immunoregulatory macrophages in the lung.
    Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 06/2011; 31(6):485-91. DOI:10.1089/jir.2011.0027 · 2.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC(50) = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC(50) = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This "double-clamp" binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.
    PLoS ONE 01/2012; 7(1):e29514. DOI:10.1371/journal.pone.0029514 · 3.23 Impact Factor
Show more