Horse heart cytochrome c entrapped into the hydrated liquid-crystalline phases of phytantriol: X-ray diffraction, Raman spectroscopic characterization

摘要

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413nm excitation, and non-resonance Raman technique with 785nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q ~(230) and Q ~(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q ~(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q ~(230) phase at 1wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568cm ~(-1) (γ _(21)) and ν _(48) mode at 633cm ~(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q ~(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature.