Bottom-Up Model Suggesting Membrane Localization of Heme Oxygenase‑1 Contributes to Heme Selection for Degradation.

Heme functions as a prosthetic group in numerous hemoproteins, whereas a fraction of intracellular heme is not tightly bound to proteins or membranes and can readily dissociate; this labile heme participates in the regulation of diverse cellular processes. Heme has pro-oxidant properties and is cytotoxic at elevated levels. Accordingly, heme oxygenase-1 (HO-1), an enzyme localized to the endoplasmic reticulum membrane, degrades excess intracellular heme. However, how HO-1 gains access to the excess intracellular heme for degradation remains unclear. In this study, we investigated how the membrane association of HO-1 influences heme capture using a bottom-up approach. A rat HO-1-based fluorescent probe retaining the membrane-anchoring region and capable of detecting heme binding via fluorescence quenching was reconstituted into liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). An HO-1 variant lacking the membrane-anchoring region competitively inhibited heme binding to the probe in the presence of POPC liposomes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein proposed to function as a heme chaperone. In contrast, membrane-associated HO-1 showed enhanced fluorescence quenching associated with heme binding in the presence of liposomes compared with aqueous conditions, although competition with GAPDH was still observed. These findings suggest that membrane environments may facilitate the access of membrane-associated HO-1 to chaperone-unbound heme under the reconstituted conditions. Our results suggest a model in which membrane localization of HO-1 contributes to the spatial regulation of intracellular heme processing and heme selection for degradation.