Characterization of molecular mechanism of neuroglobin binding to cytochrome c: A surface plasmon resonance and isothermal titration calorimetry study

Neuroglobin (Ngb) is a six-coordinate heme protein predominantly expressed in the brain tissue with a protective role against hypoxic/ischemic and oxidative stress-related insults. Several intracellular proteins including cytochrome c have been identified as potential neuroglobin binding partners. To understand the factors that control Ngb association to cytochrome c (Cyt c) the thermodynamic parameters for Cyt c association to Ngb variants were probed using surface plasmon resonance and isothermal titration calorimetry techniques. The dissociation constant for human Ngb binding to Cyt c was determined to be ~10μM using SPR and a similar value was obtained by ITC confirming that the covalent attachment of Cyt c to the gold surface modified with mixed alkanethiols does not impair Cyt c interactions with Ngb. Modification of the heme iron coordination sphere by replacement of histidine 64 in the position of sixth axial ligand with cyanide effectively inhibits the inter-protein complex formation. On the other hand, the presence of the Cys 46/Cys 55 disulfide bridge that restrains the CD loop flexibility and increases the distal histidine affinity for heme iron has no impact on complex stability. ITC data also show that the inter-protein complex formation is entropy driven (ΔH=3.3±0.2kcalmol−1 and ΔS=32.3calmol−1K−1) likely due to a reorganization of solvent molecules surrounding charged amino-acid residues. The experimental data are consistent with the results of a docking study in which the residues in the E- and F-helix of Ngb were identified to form a binding site for Cyt c. We propose that association of the exogenous ligand to the heme iron triggers a repositioning of the E-helix that prevents Ngb-Cyt c complex formation.