title: Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme.
abstract: 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.
authors: †Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48019, United States.
authors: Ireena
authors: I
authors: Bagai
authors: ‡Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United States.
authors: Ritimukta
authors: R
authors: Sarangi
authors: †Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48019, United States.
authors: Angela S
authors: AS
authors: Fleischhacker
authors: §Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.
authors: Ajay
authors: A
authors: Sharma
authors: §Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.
authors: Brian M
authors: BM
authors: Hoffman
authors: †Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48019, United States.
authors: Erik R P
authors: ER
authors: Zuiderweg
authors: †Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48019, United States.
authors: Stephen W
authors: SW
authors: Ragsdale
mesh_terms: D000595:Amino Acid Sequence; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D006418:Heme; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2