The C‑Terminal Heme Regulatory Motifs of Heme Oxygenase‑2 Are Redox-Regulated Heme Binding Sites

Heme oxygenase-2 (HO2), an enzyme that catalyzes the conversion of heme to biliverdin, contains three heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1–288 (HO2_<i>sol</i>) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2_<i>sol</i> via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe³⁺-heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of heme to disulfide-reduced HO2_<i>sol</i>, in the work described here, we further investigated the properties of Fe³⁺-heme bound to HO2. Specifically, we investigated binding of Fe³⁺-heme to a truncated form of soluble HO2 (residues 213–288; HO2_<i>tail</i>) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2_<i>tail</i> in the disulfide-reduced state binds Fe³⁺-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2_<i>sol</i> that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2_<i>sol</i> binds one Fe³⁺-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2_<i>sol</i> binds slightly more than two. Both Cys265 and Cys282 were identified as Fe³⁺-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe³⁺-heme binds with a much weaker affinity to Cys282 than to Cys265, which has an affinity much weaker than that of the His45 binding site in the catalytic core. In summary, disulfide-reduced HO2 has multiple binding sites with varying affinities for Fe³⁺-heme

Spectroscopic Studies Reveal That the Heme Regulatory Motifs of Heme Oxygenase‑2 Are Dynamically Disordered and Exhibit Redox-Dependent Interaction with Heme

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

Does Activation of the Anti Proton, Rather than Concertedness, Determine the Stereochemistry of Base-Catalyzed 1,2-Elimination Reactions? Anti Stereospecificity in E1cB Eliminations of β-3-Trifluoromethylphenoxy Esters, Thioesters, and Ketones

As part of a comprehensive investigation on the stereochemical aspects of base-catalyzed 1,2-elimination reactions, we have studied a set of acyclic carbonyl substrates that react by an irreversible E1cB mechanism with largely anti stereospecificity. ²H NMR data show that these reactions using KOH in EtOH/H₂O under non-ion-pairing conditions produce a minimum of 85–89% anti elimination on stereospecifically labeled <i>tert</i>-butyl (2<i>R</i>*,3<i>R</i>*)- and (2<i>R</i>*,3<i>S</i>*)-3-(3-trifluoromethylphenoxy)-2,3-²<i>H</i>₂-butanoate, <i>S</i>-<i>tert</i>-butyl (2<i>R</i>*,3<i>R</i>*)- and (2<i>R</i>*,3<i>S</i>*)-3-(3-trifluoromethylphenoxy)-2,3-²<i>H</i>₂-butanethioate, and the related ketones, (4<i>R</i>*,5<i>R</i>*)- and (4<i>R</i>*,5<i>S</i>*)-5-(3-trifluoromethylphenoxy)-4,5-²<i>H</i>₂-3-hexanone. With both diastereomers of each substrate available, the KIEs can be calculated and the innate stereoselectivities determined. The elimination reactions of the β-3-trifluoromethylphenoxy substrates occur by E1cB mechanisms with diffusionally equilibrated enolate-anion intermediates. Thus, it is clear that anti elimination does not depend solely upon concerted E2 mechanisms. Negative hyperconjugation provides a satisfactory explanation for the anti stereospecificity exhibited by our carbonyl substrates, where the leaving group activates the anti proton, leading to the enolate intermediate. The activation of the anti proton by negative hyperconjugation may also play a role in the concerted pathways of E2 mechanisms. We have also measured the rates of the hydroxide-catalyzed elimination reactions of butanoate, thiobutanoate, and ketone substrates in EtOH/H₂O, with β-tosyloxy, acetoxy, and 3-trifluoromethylphenoxy nucleofuges