Molecular Imaging of Labile Heme in Living Cells Using a Small Molecule Fluorescent Probe

Abstract

Labile heme (LH) is a complex of Fe­(II) and protoporphyrin IX, an essential signaling molecule in various biological systems. Most of the subcellular dynamics of LH remain unclear because of the lack of efficient chemical tools for detecting LH in cells. Here, we report an activity-based fluorescence probe that can monitor the fluctuations of LH in biological events. H-FluNox is a selective fluorescent probe that senses LH using biomimetic N-oxide deoxygenation to trigger fluorescence. The selectivity of H-FluNox to LH is >100-fold against Fe­(II), enabling the discrimination of LH from the labile Fe­(II) pool in living cells. The probe can detect the acute release of LH upon NO stimulation and the accumulation of LH by inhibiting the heme exporter. In addition, imaging studies using the probe revealed a partial heme-export activity of the ATP-binding cassette subfamily G member 2 (ABCG2), potential LH pooling ability of G-quadruplex, and involvement of LH in ferroptosis. The successful use of H-FluNox in identifying fluctuations of LH in living cells offers opportunities for studying the physiology and pathophysiology of LH in living systems.

Ferroptosis and its role in skeletal muscle diseases

“…These results suggest that the heme content in cancer cells may affect their sensitivity to ferroptosis. In addition, as another important executor of heme export, part of ABCG2's heme output capacity was found to be related to ferroptosis in recent studies (Desuzinges-Mandon et al, 2010;Kawai et al, 2022). But there is still no substantive research to support this finding yet.…”

Recent advances in small-molecule fluorescent probes for studying ferroptosis

“…27(A)). 169 Compared with probes 39 and 40 , probe 37 showed remarkable fluorescence enhancement in response to LH, and thus the later was selected as the candidate probe. Probe 37 comprised of a rhodol platform bearing an electron-withdrawing 4,4-difluoropiperidine N -oxide moiety, which served as the LH reactive site.…”

Recent Advances of Fluorescence Probes for Imaging of Ferroptosis Process

“…By contrast, the addit Fe 2+ (10 μM) resulted in only a less 10−fold fluorescence increase even after incubati 30 min. Besides, Fe 3+ and hemin (in the absence of GSH) would not affect the detect Interestingly, based on the above−mentioned N−oxide deoxygenation strategy, Hirayama et al [43] further reported a selective fluorescence probe H−FluNox for intracellular labile heme (i.e., complex of Fe 2+ and protoporphyrin IX, an essential protein cofactor), as well as its applications in ferroptosis process. The design of H−FluNox was inspired by the biomimetic reaction of cytochrome P450 with tetramesitylporphyrin iron complex and perbenzoic acid derivatives (Figure 4A) [44], in which the cleavage of O−O bond was accelerated by the electron−withdrawing aryl group.…”