Fig. 1: ROMO1 is able to scavenge ROS and form reversible intermolecular disulfides, protecting protein cysteines against irreversible oxidations.

a Electron micrographs of mitochondria in HEK293T cells, either untransfected or stably expressing ROMO1-APEX2 or APEX2-ROMO1 fusion proteins. Note that APEX2-related dark staining (white arrowheads) was confined to the inner membrane space (IMS), indicating that both the N- and C-termini of ROMO1 face the IMS. Scale bar, 200 nm. Images are representative of ≥9 fields of view from 3 independent experiments. b ROMO1 structure predicted using AlphaFold Protein Structure Database (AF-P60602-F1) and the human gene sequence. The four cysteine residues are highlighted in red. Produced with PyMOL (www.pymol.org). c In vitro H₂O₂ detoxification assay. Purified human wild type (WT) ROMO1, ROMO1-FLAG, and mutant ROMO1-FLAG in which four cysteines were converted to serine (4CS), were used at concentrations ranging from 0 to 2 μM, with the addition of 2 μM H₂O₂. Data are mean ± s.e.m. n = 7, 4, 5 measurements from 3 independent experiments for 0 μM, 1 μM, and 2 μM ROMO1 (WT), respectively; n = 12, 8, 9, 8, 8 measurements from 3 independent experiments for 0 μM, 0.2 μM, 0.5 μM, 1 μM, and 2 μM ROMO1-FLAG (WT), respectively; n = 8, 11, 5, 5, 3 measurements from 3 independent experiments for 0 μM, 0.2 μM, 0.5 μM, 1 μM, and 2 μM ROMO1-FLAG (4CS), respectively. Note that ROMO1 and ROMO1-FLAG show similar H₂O₂ detoxifying activity. d Kinetic analysis of GSH-reducing system-mediated reduction of pre-oxidized ROMO1-FLAG. The reduction of pre-oxidized ROMO1-FLAG was assessed using a coupled spectrophotometric assay. The inset shows a plot of the initial NADPH consumption rates against different concentrations of pre-oxidized ROMO1-FLAG. A rate constant (k = 2.0 × 10³M^-1s^-1) was determined. Data are mean ± s.d. n = 3, 3, 5, 3, 3, 3 measurements from 3 independent experiments for 0 μM, 0.5 μM, 1 μM, 5 μM, 7.5 μM, and 10 μM pre-oxidized ROMO1-FLAG, respectively. e Kinetic analysis of TRX2-reducing system-mediated reduction of pre-oxidized ROMO1-FLAG using coupled spectrophotometric assay. The inset shows a plot of initial NADPH rates against different concentrations of pre-oxidized ROMO1-FLAG. Apparent K_m (K_{m, app} = 9.8 × 10^-6M) and apparent V_max (V_{max, app} = 653 nM^-1min^-1) were determined. Data are mean ± s.d. n = 3 measurements from 3 independent experiments for different concentrations of pre-oxidized ROMO1-FLAG. f Non-reducing SDS-PAGE analysis for disulfide-bonded homodimer formation of ROMO1 and its sensitivity to TRX2- and GSH-reducing systems. ROMO1-FLAG protein was pre-oxidized with 10 µM H₂O₂. The ROMO1-FLAG homodimer is indicated by the red asterisk. GR, glutathione reductase. TrxR, thioredoxin reductase. Coomassie brilliant blue (CBB) R-250 staining was used. g Changes of aconitase activity in young WT and TG cardiac or liver mitochondria following H₂O₂ treatment. Heart and liver mitochondria were exposed to 50 and 200 μM H₂O₂, respectively. Data are mean ± s.e.m. For heart mitochondria, n = 8 mice (4 M 4 F) in WT-Basal, n = 6 mice (3 M 3 F) in TG-Basal, n = 6 mice (3 M 3 F) in WT-H₂O₂, n = 5 mice (2 M 3 F) in TG-H₂O₂; for liver mitochondria, n = 6 mice (3 M 3 F) in WT-Basal, n = 6 mice (3 M 3 F) in TG-Basal, n = 6 mice (3 M 3 F) in WT-H₂O₂, n = 7 mice (4 M 3 F) in TG-H₂O₂. 3-month-old WT and TG mice were used. h Anti-ROMO1 western blots showing DTT-sensitive high molecular weight complexes in neonatal rat cardiomyocytes. Note that their formation was promoted by ROMO1-FLAG overexpression and H₂O₂ treatment (200 μM). Anti-ATP5B served as the internal control. i In vitro intermolecular disulfide formation assay using purified ROMO1-FLAG and SDH complex. SDHA and ROMO1 formed a complex (red asterisk) after 100 μM H₂O₂ treatment, which was sensitive to DTT (10 mM). CBB staining was used. j Reduction of the intermolecular disulfide between ROMO1 and SDHA by TRX2- and GSH-reducing systems. Red asterisk indicates the ROMO1-SDHA complex. CBB staining was used. k Overexpressing ROMO1-FLAG alleviated S-sulfinic levels induced by 200 μM H₂O₂ treatment in neonatal rat cardiomyocytes. A diazene-based alkyne probe was used to label S-sulfinylated proteins. CBB staining served as the internal control. Data in f, h–k are representative of three independent experiments. In g, two-way ANOVA with Tukey’s multiple comparisons test was used. *p < 0.05, ***p < 0.001. Source data are provided as a Source Data file.