Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions

Abstract

Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site. Outside their catalytic cores are two regions unique to HO2: a 20-amino acid–long N-terminal extension and a C-terminal domain containing two heme regulatory motifs (HRMs) that bind heme independently of the core. Both HO isoforms contain a C-terminal hydrophobic membrane anchor; however, their sequences diverge. Here, using hydrogen–deuterium exchange MS, size-exclusion chromatography, and sedimentation velocity, we investigated how these divergent regions impact the dynamics and structure of the apo and heme-bound forms of HO1 and HO2. Our results reveal that heme binding to the catalytic cores of HO1 and HO2 causes similar dynamic and structural changes in regions (proximal, distal, and A6 helices) within and linked to the heme pocket. We observed that full-length HO2 is more dynamic than truncated forms lacking the membrane-anchoring region, despite sharing the same steady-state activity and heme-binding properties. In contrast, the membrane anchor of HO1 did not influence its dynamics. Furthermore, although residues within the HRM domain facilitated HO2 dimerization, neither the HRM region nor the N-terminal extension appeared to affect HO2 dynamics. In summary, our results highlight significant dynamic and structural differences between HO2 and HO1 and indicate that their dissimilar C-terminal regions play a major role in controlling the structural dynamics of these two proteins.

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

“…Because we compared the H45W/G159W variant to itself (not to wild-type protein), changes in exchange were strictly due to the presence of heme, not the mutations themselves. Further, the changes observed with the H45W/G159W variant are quite distinct from those of previous observations in which heme was in the core (20). In those experiments, no difference in uptake was observed between wild-type Fe 3+ core/HRM-HO2sol R relative to wild-type Fe 3+…”

“…Lastly, a significant level of protection in the area around residue 100 was observed in the H45W/G159W variant. We have previously noted protection from exchange in this region upon heme binding to the core of HO2, when protection is also observed in the distal helix (20), suggesting that the two regions interact in some way. Thus, overall, the H45W/G159W variant appears to have a similar global structure as wild-type HO2sol, with the substitutions having introduced some rigidity into the heme binding pocket.…”

“…1). A subsequent study using hydrogen-deuterium exchange mass spectrometry (HDX-MS) did not uncover any evidence of docking when both the core and the HRMs were fully occupied with Fe 3+ -heme (Fe 3+ core/HRM-HO2 R ) (20). To fully examine our hypothesis that the tail participates in heme transfer to the catalytic core, it was necessary to examine a construct designed to have a heme-loaded tail and an apo core (Fe 3+…”

Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

“…In HO2, one KFERQ-like motif, 52[QFVKD]57, absent in HO1, is found at the proximal helix, which harbors the heme ligand His45 at the HO2 catalytic site. According to our HDX-MS analysis, compared with an apo-active site, decreased deuterium incorporation at the proximal helix was observed when heme is bound at the catalytic site (45). This heme-dependent protection of the KFERQ-like motif could explain how the apo active site of HO2 is differentially recognized by the lysosomal degradation machinery, leading to more rapid protein degradation.…”

“…This finding clearly shows that the degradation signal is not triggered by changes in the levels of the products (CO, Fe, biliverdin) of heme degradation. Our recent hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies demonstrated that in H45W/G159W variant, the tryptophan mutations themselves led to minimal to no structural perturbations and that the only observed differences in HDX-MS related to heme binding to the catalytic core were specific to the distal and proximal helices, which form the heme binding pocket (45). Furthermore, the circular dichroism spectra of wild-type HO2 and the H45W/G159W variant were nearly indistinguishable (30).…”