Redox-linked conformational changes in proteins detected by a combination of infrared spectroscopy and protein electrochemistry. Evaluation of the technique with cytochrome c.
We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c, because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reduced-minus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-1 and 1800 cm-1.
Moss
D
D
Institut für Biophysik und Strahlenbiologie, Universität Freiburg, Federal Republic of Germany.
Nabedryk
E
E
Breton
J
J
Mäntele
W
W
D000818:Animals; D003574:Cytochrome c Group / Q000592:standards; D004563:Electrochemistry / Q000295:instrumentation; D004566:Electrodes; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011506:Proteins; D012015:Reference Standards; D013055:Spectrophotometry, Infrared; D014961:X-Ray Diffraction
D003574:Cytochrome c Group; D011506:Proteins