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Redox-linked structural changes in cytochrome C probed by stationary and time-resolved surface enhanced infrared absorption spectroscopy |
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| รหัสดีโอไอ | |
| Title | Redox-linked structural changes in cytochrome C probed by stationary and time-resolved surface enhanced infrared absorption spectroscopy |
| Creator | Nattawadee Wisitruangsakul |
| Contributor | Sanong Ekgasit, Zebger, Ingo |
| Publisher | Chulalongkorn University |
| Publication Year | 2551 |
| Keyword | Infrared spectroscopy, Cytochrome c, Seira, อินฟราเรดสเปกโทรสโกปี, ไซโตโครมซี |
| Abstract | The application of surface enhanced infrared absorption (SEIRA) spectro-electrochemistry for studying an immobilized protein yields advantages with respect to its surface sensitivity and probing the whole protein backbone. For an electrostatic immobilization, cytochrome c (Cyt-c) was adsorbed on a Au electrode via a binding to self-assembled monolayers (SAMs) of variable-length ω-carboxyl alkanethiols. In this work, stationary SEIRA measurements were performed to elucidate the structural changes of the SAMs, the redox-linked structural and orientational changes of the immobilized Cyt-c, and to study the non-native B2 state of Cyt-c. For the first time, the application of time-resolved SEIRA spectroscopy was reported to probe the protein dynamics coupled with the redox processes of Cyt-c. Potential dependent spectral changes of the SAMs revealed only reversible variations within the potential range of interest (-0.1 to +0.2 V), which do not interfere with the investigation of Cyt-c bound to these SAMs. However, depending on the SAM-lengths, irreversible spectral changes could be found also at certain potentials outside the mentioned range. The -turn III (1673 cm-1) and -turn II/ -helix (1660 cm-1) segments of ferric Cyt-c revealed orientational changes with respect to the surface under the influence of the electric field strengths, which were found to be correlated with the conformational transition from the native B1 to the non-native B2 state. The potential dependent spectra of the B2 conformation obviously differ from that of the B1 state. It reveals broad complex bands caused by an alteration of the tertiary structure and an arrangement of the hydrogen-bonding interaction involving the movement of peptide backbone of -turn II/ -helix (ca. 1659 cm-1) and unordered (ca. 1645 cm-1) structures for the formation of non-native B2 conformation. The redox potentials of the B1 state determined by stationary SEIRA spectroscopy and cyclic voltammetry (CV) are not significantly different from that in solution. CV measurements demonstrate a reversible electron transfer (ET) process and a non-exponential distance-dependence of the ET rate. On the basis of characteristic of redox state sensitive amide I bands, the protein structural changes triggered by the ET are monitored by rapid scan and step scan SEIRA spectroscopy in combination with the potential jump technique. Whereas the temporal evolution of the conjugate bands at 1693 and 1673 cm-1 displays the same rate constants as ET rate, the time-dependent changes of the 1660-cm-1 band are slower by about a factor of 2. The study demonstrates that time-resolved SEIRA spectroscopy provides additional information about the dynamics and mechanism of interfacial processes of redox proteins, thereby complementing the results obtained from other surface-sensitive techniques. |
| URL Website | cuir.car.chula.ac.th |
