FIGURE LEGENDS
Figure 1. (A ) Schematic diagram of Ncb5or constructs. Except Ncb5or-Δ34, all are used in the current study. (B ) Amino acid sequences of the N-terminal region in human Ncb5or and its point-mutation variant.
Figure 2. Far-UV (A ) and near-UV/visible (B ) CD spectra of Ncb5or-N/b5 and Ncb5or-b5. Far UV (C ) and near-UV/visible (D ) spectra of Ncb5or-N as a stand-alone peptide (alone) or when fused to Ncb5or-b5 (difference, N/b5 – b5). Difference far UV (E, G ) and near-UV/visible (F, H ) CD spectra of Ncb5or-N/b5 variants. Difference spectra were presented by smoothing raw data, which are shown in Figure S3 , with the use of negative exponential program in SigmaPlot 10. Molar ellipticity is used in all CD spectra. Values of signature bands are summarized in Table 1 .
Figure 3. (A ) Comparison of the domain arrangements in Ncb5or and RLF. (B ) Amino acid sequence alignment of human Ncb5or (residues 1 – 137), rice RLF (Oryza sativa, residues 72 – 218), and baker’s yeast IRC21 (Saccharomyces cerevisiae, residues 17-201). Identical and similar residues are coded in yellow and green, respectively.
Figure 4. (A) Structure of RLF (subunit A) with the secondary structure elements annotated. The heme molecule is rendered as gold cylinders. (B) RLF sequence with the secondary structure elements annotated. The red arrows indicate the His residues that coordinate the heme.
Figure 5. Superposition of rice RLF (magenta) with the Ncb5or b5-domain (PDB 3LF5, green). RMSD deviation between Ca for b5 core residues (see Figure 4) is 1.01 Å.
Figure 6. (A) Comparison of conserved, surface exposed tryptophan residue in Ncb5or (green) and RLF (magenta). Trp199 in the RLF structure forms a hydrogen bond with a heme propionate group, while Trp114 in Ncb5or extends into solvent. This flexibility suggests a possible role in modulating electron flow. (B) Comparison of the heme orientation and the coordinating His residues. His89/His112 are colored green for 3LF5 and His174/His197 are colored magenta for RLF.
Figure 7. View of the RLF structure highlighting helix α1 and locations/interactions of residues. Corresponding Ncb5or residues are shown in parentheses. Black dotted lines indicate hydrogen bonds.
Figure 8. Residue Trp120 in RLF, corresponding to Trp37 in Ncb5or, forms a network of hydrophobic packing interactions with other conserved residues (shown with carbon atoms rendered in yellow). The hydroxyl group of Tyr170 (Tyr85 in Ncb5or) makes van der Waals contact with the heme, and the thiol group of Cys209 (Cys124 in Ncb5or) makes van der Waals contact with Trp120.
Figure S1. Sequence alignment of N-terminal region and cytochrome b5 domain of known Ncb5or proteins of a diverse background. Asterisk and “_” indicate conserved and chemically similar residues , among mammals, respectively. Non-conserved residues at key positions are indicated in red. Species (GenBank accession number) are as follows: (mammals in yellow) human (NM_016230), monkey (XP_518614.3), monkey Old World (XP_011920356.1), guinea pig (XP_005006001), mouse (NM_024195), rat (NM_133427), cow (NM_001038159), horse (XM_001499913), sheep (XP_027818354), pig (XP_013848110), dog (XM_532219), minke whale (XM_007165020.1), ferret (XP_004773650), bat/Molossus (XP_036104100), bat/Pterpus (XP_011363184), brown bat (XM_008145048), opossum (XM1375853), platpus (XP_028903113); (birds, reptiles and amphibians in green) Zebra Finch (XP_030123394), golden eagle (XP_029856007), peregrine falcon (XP_013152060), chicken (XM_001233870), Dalmatian pelican (KFQ624870), crested ibis (KFR09350), seriema (KFP68580), swan goose (XM_013180133), turkey (XP_010705963), turtle/Terrapene (XP_026510156) turtle/Pelodiscus (XP_006133410), garter snake (XP_032072321), Xenopus (XP_031757575), Coelacanth (XP_014343350); (fishes in blue) Cyprinidae (XM_016550377), Zebrafish (NP_001018496), Pimephales (XP_039507272), Salmon (XP_020361560), bony fish (XM_008279520), yellow croaker (XM_019265374), Fugu (TNN02071), Pufferfish/Japan (XM_003965751), tire track eel (XR_003294861), swamp eel (XM_020621659), Gasterosteus (XP_040020873), rice fish (AM140533), rice fish India (XP_024117733), Lamprey (XP_032829495), Skate (XP_032877801), seahorse (XM_019880106), (invertebrates in grey) Lancelet (XM_002603916), Trichoplax (XM_002112883), tunicate (XR_053035), Hydra (XM_002165807), sea anemone (XP_048577331), coral (EZ038676, Acropora), coral/Exaiptasia (KXJ05517), sea urchin/Lytechinus (XP_041463305), sea urchin (XP1198870), Teladorsagia (PIO73582), hookworm/Necator (RCN51358), C.elegans (NM_001026613), pig worm (KFD50571c, Trichuris suis), Trichuris (KHJ45328); (insects in purple) Drosophila (NM_137575), Mosquito/Anopheles (XP_041781397), Mosquito/Aedes (KXJ83737), Honeybee (XM_394412), Louse (XM_002428283), Jewel wasp (XM_001601866), Beetle (XM_963135), Pea aphid (XM_001948299), Deer tick (XM_002401084),
Figure S2. Prediction of disordered domains in Ncb5or by PONDR VL-XT (A ). Prediction of secondary structure in the 50 N-terminal residues of Ncb5or by PSIPRED and PROF (B ) and by AGADIR (C ). AA (amino acid): Target sequence, Pred: Predicted secondary structure (H=helix, E=strand, C=coil), Conf: Confidence (0=low, 9=high).
Figure S3. Sequence alignment of the N-terminal region and b5 core (partial) of human Ncb5or against RLF proteins in plants. Asterisk (*) indicates conserved residues, whereas “_” represents chemically similar residues.
Figure S4 . Sequence alignment of the N-terminal region of human Ncb5or against IRC21 proteins in fungi. Asterisk (*) indicates conserved residues, whereas “_” represents chemically similar residues.
Figure S5 . Sequence alignment of the N-terminal region and cytochrome b5 domain of human Ncb5or against cytochrome b5 proteins in protists (Dictyostelium and Trypanosoma ) and parasites (all others). Asterisk (*) indicates conserved residues, whereas “;” represents chemically similar residues. The conserved “HPGG” heme binding site is highlighted, and the N-terminal motif in human and protists is indicated by “——“.