Cytoglobin Interactions with Hydrophobic Probe 1,8-ANS

Abstract

Cytoglobin (Cygb) is small heme protein that belongs to the hexa-coordinate hemoglobin family. Cygb is over-expressed in fibrosis and neurodegenerative disorders, whereas it is down-regulated in some types of cancer including head and neck cancer. A recent report indicates that Cygb binds sodium oleate, which promotes conformational transition from hexa to penta-coordinated heme iron suggesting a possible role of Cygb in lipid signaling. To understand the mechanism of Cygb-lipids interactions, we have characterized the interactions between the hydrophobic probe 1-anilino-8-naphthalene sulfonate (1,8-ANS) and Cygb in the presence and absence of sodium oleate using steady-state and time-resolved fluorescence spectroscopy and isothermal titration calorimetry. Cygb binds 1,8-ANS in the ferric, ferrous and exogenous ligand bound form. Addition of sodium oleate to Cygb-ANS complex leads to a decrease in the 1,8-ANS emission intensity indicating competition between the lipid and ANS for Cygb binding sites. Two binding sites were identified using ITC for ANS binding to Cygb, one with moderate affinity (Kd ∼ 50 μM) and a low affinity binding site (Kd ∼ 2 mM). Reduction of the internal disulfide bond in Cygb slightly decreases the affinity of 1,8-ANS to Cygb, whereas binding of cyanide results in a 1.5-fold decrease in affinity of Cygb for the hydrophobic probe (Kd=76 μM). Molecular docking studies of 1,8-ANS with Cygb were performed and the data suggest that 1,8-ANS binds to the extended termini in Cygb.