The C-terminal heme regulatory motifs of heme oxygenase-2 are redox-regulated heme binding sites

Abstract

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 (HO2sol) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2sol via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe(3+)-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 HO2sol, in the work described here, we further investigated the properties of Fe(3+)-heme bound to HO2. Specifically, we investigated binding of Fe(3+)-heme to a truncated form of soluble HO2 (residues 213-288; HO2tail) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2tail in the disulfide-reduced state binds Fe(3+)-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2sol that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2sol binds one Fe(3+)-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2sol binds slightly more than two. Both Cys265 and Cys282 were identified as Fe(3+)-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe(3+)-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 Fe(3+)-heme.

Figure 1

Characterization of HO2 in the Fe³⁺-heme-bound forms. (A) Absorbance spectra of 5 μM Fe³⁺-HO2_core (…), Fe³⁺-HO2_tail^R (---), or Fe³⁺-HO2_sol^R (—) in 50 mM Tris (pH 8.0) and 50 mM KCl at 20 °C. (B) EPR spectra of Fe³⁺-HO2_core, Fe³⁺-HO2_tail^R, Fe³⁺-HO2_sol^R, and Fe³⁺-HO2_sol(H45A)^R. Samples were prepared and run as described (see Methods). The g values are indicated above the spectra.

Figure 2

N and N DAVIES ENDOR spectra of unlabeled and N-labeled Fe³⁺-HO2 measured at the indicated g values. Spectra were recorded of N-labeled Fe³⁺-HO2_sol^R (red), N-labeled Fe³⁺-HO2_sol^O (purple), unlabeled Fe³⁺-HO2_sol^R (black), unlabeled Fe³⁺-HO2_tail^R (blue), and N-labeled Fe³⁺-HO2_sol^R (green). Experimental conditions: MW frequency of 34.7 GHz, T of 2 K, pulse sequence described in Methods, tp of 40 ns, τ of 600 ns, trf of 30 μs, trep of 50 ms, and 256 transients/scan.

Figure 3

Absorbance spectra of wild-type Fe³⁺-HO2_tail^R and its variants. Spectra were recorded at 20 °C in 50 mM Tris (pH 8.0) and 50 mM KCl with a protein concentration of 5 μM. (A) Wild-type Fe³⁺-HO2_tail^R (—), Fe³⁺-HO2_tail(C265A)^R (…), and Fe³⁺-HO2_tail(C282A)^R (---). (B) Fe³⁺-HO2_tail(C282A)^R (---) and Fe³⁺-HO2_tail(H256A/C282A)^R (·-·). (C) Fe³⁺-HO2_tail(C265A)^R (---) and Fe³⁺-HO2_tail(H256A/C265A)^R (·-·).

Figure 4

EPR spectra of Fe³⁺-HO2_tail^R variants. Samples were prepared and run as described (see Methods). The g values are indicated above the spectra of Fe³⁺-HO2_tail(C265A)^R, Fe³⁺-HO2_tail(C282A)^R, Fe³⁺-HO2_tail(H256A/C265A)^R, and Fe³⁺-HO2_tail(H256A/C282A)^R.

Figure 5

Temperature dependence of Fe³⁺-HO2_tail^R. The absorbance spectrum of 5 μM Fe³⁺-HO2_tail^R in 50 mM Tris (pH 8.0) and 50 mM KCl was recorded at 35 (---), 20 (…), and 5 °C (—).

Figure 6

Fe³⁺-heme difference titrations with HO2_core, HO2_tail(C282A)^R, HO2_tail(C265A)^R, and HO2_sol^R. Difference titrations were performed in 50 mM Tris (pH 8.0) and 50 mM KCl at 20 °C under anaerobic conditions with (A) 2 μM HO2_core, (B) 10 μM HO2_tail(C282A)^R, (C) 6 μM HO2_tail(C265A)^R, or (D) 5 μM HO2_sol^R. Insets in panels A–C are plots of the respective data at the indicated wavelength with the solid lines representing the fits to the quadratic equation to obtain K_d values.

Figure 7

Plot of observed rate constants as a function of the concentration of available Fe³⁺-heme binding sites on HO2. The apo form of HO2_core (▼), HO2_tail(C282A)^R (●), or HO2_sol^R (◆) was mixed at various concentrations with Fe³⁺-heme in a stopped-flow instrument within an anaerobic chamber. Data were collected at 20 °C while monitoring was conducted at 406 nm. The data were fit to eq 1 as described in the text, and the resulting fits are represented as solid lines.

Scheme 1

Binding of Fe³⁺-Heme to HO2