Figure 1

Characterization of iron–sulfur clusters in as-isolated clMagR. (a) Sequence alignment of MagR in eight representative species. Predicted secondary structures are shown in the upper lines, with two alpha-helices (orange cylinders) and seven beta-strands (green arrows). Conserved residues with iron–sulfur cluster binding properties are shown in the red background (100% conserved), indicated by stars. Other conserved residues are shown in the gray background and bold fonts. Species’ common name, Latin name and sequence ID in NCBI are listed as follows: Pigeon (Columba livia), XP_005508102.1*; Zebra finch(Taeniopygia guttata), XP_002194930.1*; Fly(Drosophila melanogaster), NP_573062.1*; Monarch butterfly(Danaus plexippus), AVZ24723.1*; Salmon(Salmo salar), XP_013999046.1*; Octopus(Octopus bimaculoides), XP_014786756.1*; Little brown bat(Myotis lucifugus), XP_006102189.1*; Human(Homo sapiens), NP_112202.2*. (b) UV–Vis absorption spectrum of as-isolated pigeon MagR (clMagR^WT, black) and C60AC124AC126A substitution mutant (clMagR^3M, red), indicating three cysteines contribute to the iron–sulfur cluster binding. SDS-PAGEs of protein preparation are shown as inserts, theoretical mass of the clMagR monomer and clMagR^3M monomer were 16.41 kDa, 16.31 kDa, respectively. (c) CD spectrum of as-isolated clMagR^WT(black) and clMagR^3M(red). (d, e) X-band EPR spectrum of as-isolated clMagR^WT at oxidized (d) and reduced status (e). The samples were frozen in TBS buffer and the spectrums were recorded at various temperatures (10 K, 25 K, 45 K, 60 K). (f) Low-temperature resonance Raman spectra of as-isolated clMagR^WT. Spectra were recorded at 17 K using 488 nm laser excitation.