Fig. 6

CrCYB5D1 binds heme in a redox-sensitive manner. (A) Comparison of the heme concentration in flagella of HS211 and Crcyb5d1. All values are shown as the mean ± SD of four independent experiments. n.s., not significant. Immunoblot analysis of the binding activity of CrCYB5D1::Strep from whole-cell lysate (B) or freshly purified CrCYB5D1::Strep (C) from HS211 cells expressing CrCYB5D1::Strep using hemin-conjugated agarose and an anti-Strep antibody (Upper); IFT46::YFP is a negative control in B. In C, the total protein was assessed by Coomassie blue staining (CBB, Lower). ?Pre,? the soluble fraction of the cell lysate before incubation with hemin-conjugated agarose beads; ?Post,? the soluble fraction after incubation with hemin-conjugated agarose beads; ?W1,? ?W2,? and ?W3,? wash fractions; ?E,? the bound fraction eluted with SDS and ?-mercaptoethanol. (D) The absorbance spectra between 300 and 600 nm were collected for 3 ?M CrCYB5D1::Strep, 50 ?M hemin, and 50 ?M hemin mixed with 3 ?M CrCYB5D1::Strep. (E) The maximum absorbance (A_max) at 402 nm resulting from heme binding to CrCYB5D1::Strep was plotted for a concentration series of hemin mixed with 3 ?M CrCYB5D1::Strep. (F) Assessment of the redox state of purified CrCYB5D1::Strep from whole cells. 50 mM GSSG or 1 mM DTT were used as oxidative and reductive treatments, respectively; NT indicates no treatment. The quantitative ratio of reduced and oxidized CrCYB5D1 in F under different conditions (see main text) was determined by immunoblot analysis. (G) Detection of heme-promoted peroxidase activity using purified CrCYB5D1::Strep. The top shows Coomassie blue staining; the second panel indicates peroxidase activity of the CrCYB5D1?hemin complex; the third panel shows peroxidase activity with free hemin, and the bottom shows immunoblot analysis of samples probed with Strep antibody.