Infrared Spectroscopic Discrimination between the Loop and α-Helices and Determination of the Loop Diffusion Kinetics by Temperature-Jump Time-Resolved Infrared Spectroscopy for Cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the α-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70–85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I′ region indicate that the loop and the helical absorption bands overlap at 1653 cm−1 at room temperature. Thermal titration of the amide I′ intensity at 1653 cm−1 reveals that a transition in loop structural change occurs at lower temperature (Tm = 45°C), well before the global unfolding of the secondary structure (Tm ≈ 82°C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25°C to 35°C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm−1, overlap with the helical absorption) to random coil (1645 cm−1) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was ∼3.6 × 106 s−1.