Characterization of the BioB:HscA complex using equilibrium analytical ultracentrifugation. Protein samples were equilibrated in 50 mM Tris HCl (pH 7.5), 100 mM NaCl, 1 mM TCEP, centrifuged at speeds of 10,000, 12,000, and 15,000 rpm for 8 h, and radial absorbance scans were collected at 452 nm. At this wavelength, only protein species that include BioB are detected. (A) 2Fe-BioB (7.5 μM). The data are fit to a two-component model with M1 = 90,680 (90.5 % of the mixture) and M2 = 173,860. (9.5 %), corresponding to BioB2 and BioB4, respectively. (B) 2Fe-BioB (7.5 μM) and HscA (15 μM). The data are fit to a three-component model with M1 = 81,410 (50.6 %), M2 = 139,980 (33.3 %), and M3 = 217,920 (16.1 %), corresponding to BioB2, BioB2:HscA, and BioB2:HscA2 respectively.