Figure 3: GLB-12 produces O₂^.− as a signalling molecule.

(a) Ribbon representation of the GLB-12 globin domain, with helices labelled according to the canonical globin fold (green). The GLB-12-specific pre-A helix and F’-helix are highlighted in red. The terminal residues visible in the electron density (19–215) are labelled. The protein electrostatic surface (see also panel (b)) is displayed in semitransparent colours, allowing the view of underlying secondary structure elements and a distal apolar tunnel (magenta mesh) located between the B-, E- and G-helices. Acidic residues, localized where this apolar tunnel reaches the protein surface, are shown in stick representation (yellow) and labelled. (b) Electrostatic surface of the GLB-12 globin domain. The blue and red colours highlight positively and negatively charged surfaces, respectively. The haem moiety, with the propionate groups fully exposed to the solvent region, is shown as stick representation (yellow). (c) Detail of the GLB-12 haem cavity, showing the presence of two histidines and three additional polar amino acids. (d) The GLB-12 K88L mutant is less capable of O₂^.− production, while R95L and R129L mutants are incapable of O₂^.− production. (e) These three mutants, when expressed as RNAi-resistant (RR) genes, are not capable of rescuing the glb-12 RNAi reduction in fecundity (n=5). Percentages show control RNAi compared with glb-12 RNAi within a strain. *P<0.05; **P<0.01 (two-sided Student t-test). Data are mean±s.e.m.