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

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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.

heme oxygenase
enzyme catalysis
enzyme mechanism
protein dynamics
membrane protein
hydrogen exchange mass spectrometry
enzyme kinetics
conformational change
heme homeostasis
membrane anchor

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This work was supported by National Institutes of Health Grants R01-GM123513 (to S. W. R.) and P30GM103335 and GM061068 (to D. F. B.) and a research collaboration with Waters Corp. (to J. R. E.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains Figs. S1–S6 and Excel File S1.

1

Both authors contributed equally to this work.

2

Present address: Protein Mass Spectrometry, Analytical R&D, Process R&D, Merck and Co., Inc., 2000 Galloping Hill Rd., Kenilworth, NJ 07033.