Spectroscopic Studies Reveal That the Heme RegulatoryMotifs of Heme Oxygenase-2 Are Dynamically Disordered and ExhibitRedox-Dependent Interaction with Heme

摘要

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 spectroscopicexperiments with HO2 demonstrate that, when the HRMs are in the oxidizedstate (HO2^O), both the extra N-terminal and the C-terminalHRM-containing regions are disordered. However, protein NMR experimentsillustrate that, under reducing conditions, the C-terminal regiongains 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 HRMdomains. Further, electron nuclear double resonance and X-ray absorptionspectroscopic studies demonstrate that, upon reduction of the HRMsto the sulfhydryl form, a cysteine residue from the HRM region ligatesto a ferric heme. Taken together with EPR measurements, which showthe appearance of a new low-spin heme signal in reduced HO2, it appearsthat a cysteine residue(s) in the HRMs directly interacts with a secondbound heme.