Characterization of Membrane-Bound Alpha-Synuclein with the Thiocyanate Vibrational Probe Group

Abstract

The L-site binding of ferricytochrome c to cardiolipin (CL) -containing liposomes at slightly acidic pH has attracted interest owing to its potential role in converting this classical electron transfer protein into a peroxidase. Various lines of evidence presented by Nantes and coworkers suggest that this mode of binding is electrostatic, active below pH 7, and involves residues K22, K25, and K27 as well as H26 and H33. We combined several spectroscopic techniques to characterize L-site binding thermodynamically and to identify concomitant structural changes. To this end we employed and extended a recently presented thermodynamic model that describes cytochrome c binding to CL-containing liposomes as a two-step process, where native-like liposome-bound conformers convert to more unfolded conformers with an increase in CL concentration. These partially unfolded species are characterized by a loss of the M80 ligand, which is likely replaced by H26 and/or H33. At acidic pH, the partially unfolded low-spin species converts into a mixture of penta-and hexacoordinate high-spin species at moderate-to-high CL concentration. This change of the heme iron's spin state is most likely caused by the protonation of the non-native histidine ligand, which is facilitated by low effective pH at the liposome surface. Results of our analysis of binding isotherms obtained in the absence and presence of NaCl confirmed the electrostatic character of L-site binding. The inhibiting effect of sodium ions is attributed to the accumulation of cations in the liposome's double layer, which reduces its surface potential. Thus, L-site binding is clearly distinct from cytochrome c -CL interactions at neutral pH where only the conformational change on the membrane surface is affected by the presence of NaCl.