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

Abstract

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.

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

“…Oxidation of the thiol presumably involves electron transfer to the ferric heme, although ferrous heme was not detected in the system, presumably because it is aerobically reoxidized. A much slower rate of Cys 265 oxidation was observed during heme titration of the reduced A/ 13 (17,29). To determine if this ligand exchange occurs at something closer to physiological temperatures, we monitored the NMR spectrum of the oxidized A/ Tryptophan fluorescence quenching measurements of the binding affinity of the WT and mutant HO-2 proteins for heme revealed an ϳ2.5-fold difference.…”

Heme Sensor Proteins

“…Disulfide reduction to dithiol may lower active site heme affinity and increase the cellular heme pool. CP motifs may thus be nonessential for HO-2 heme binding and instead have signaling or molecular interaction functions (31)(32)(33).…”

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

“…Rather, HRM1 and HRM2 form a thiol/ redox switch that modulates HO-2 affinity, on the basis of the redox environment. Reducing conditions release the Cys265 thiol group from a disulphide bond with Cys282, allowing it to form an alternative axial heme ligand with lower affinity than His45 [66]. Thus, only one heme moiety conjugates with HO-2, coordinated by either His45 or the lower affinity Cys265.…”