Fig. 6. nduf-2.1 Cys366 mediates complex I ROS induced increase in locomotion.

a Schematic illustration of complex I (PDB:6ZR2) showing site of NDUFS2 Cys347 (mammalian ortholog of C. elegans NDUF-2.1 Cys366). b Expanded crystal structure of mammalian C347 of NDUFS2 (blue) and nearby NDUFS7 (red). c Ortholog alignment of H. sapiens, M. musculus and C. elegans position of the Cys residue. d Complex I ROS induced increase in locomotion requires nduf-2.1 Cys366. Wild-type, nuo-1::Supernova, nduf-2.1(C366S), nuo-1::Supernova + nduf-2.1(C366S), and nduf-2.1(C366D) L4 worms were singled and transferred to an unseeded plate. Body bends were scored for 15 s with and without illumination (MVX, 5.6 mW/mm²). Data are mean ± SD. N = 15 independent animals across 3 technical replicates, **p < 0.01 (Two-way ANOVA, Tukey’s multiple comparisons). e Rotenone increase in C. elegans locomotion requires nduf-2.1 Cys366. nuo-1::Supernova and nuo-1::Supernova + nduf-2.1(C366S) were treated with 1 µM rotenone for 24 h. Body bends were scored on unseeded plates. Data are mean ± SD. N = 15 independent animals across 3 technical replicates, **p < 0.01 (Two-way ANOVA, Sidak’s). Full dose response in Supplementary Fig. 1. f Complex I ROS-mediated phototaxis requires nduf-2.1 Cys366. Wild-type, nuo-1::Supernova, nduf-2.1(C366S), and nuo-1::Supernova with nduf-2.1(C366S) worms were transferred to the center of the seeded plate with half of the plate shaded (dark). Worms were then illuminated (GYX, 0.78 mW/mm²) for 120 min and the number of worms in light and dark sections were scored and the phototaxis index was calculated. Data are mean ± SD. N = 3 independent experiments, each containing 40–90 animals, **p < 0.01 (Two-way ANOVA, Tukey’s multiple comparisons). g nduf-2.1 Cys366Asp results in loss of complex I activity. Total complex I activity was measured from wild-type, nuo-1::Supernova, nduf-2.1(C366S), nuo-1::Supernova with nduf-2.1(C366S), and nduf-2.1(C366D) isolated freeze-thawed mitochondria. Data are mean ± SD. N = 12, 14, 6, 6, 7 independent mitochondrial preparations, *p < 0.05 vs WT (One-way ANOVA, Dunnett’s multiple comparisons). h Light-induced complex I inhibition requires nduf-2.1 Cys366. Isolated freeze-thawed mitochondria from wild-type, nuo-1::Supernova, nduf-2.1(C366S), and nuo-1::Supernova + nduf-2.1(C366S) worms were exposed to light (GYX, 7.8 mW/mm²) for 10 min and total complex I activity was measured and expressed as percent of no light control. Data are mean ± SD. N = 5, 10, 5, 6 independent mitochondrial preparations, *p < 0.05 vs all groups (One-way ANOVA, Tukey’s multiple comparisons).