REDOX-INDUCED CONFORMATIONAL-CHANGES IN MYOGLOBIN AND HEMOGLOBIN - ELECTROCHEMISTRY AND ULTRAVIOLET VISIBLE AND FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY AT SURFACE-MODIFIED GOLD ELECTRODES IN AN ULTRA-THIN-LAYER SPECTROELECTROCHEMICAL CELL

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (almost-equal-to 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10 000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C=O modes because of their small (2-5 cm-1) shift in (H2O)-H-2. A similar assignment could be achieved for amide II modes because of their strong shift in (H2O)-H-2. On the basis of their vibrational frequency and structural arguments, a part of these signals is attributed to alpha-helical regions of the proteins. Although the band assignment is far from being complete, the results demonstrate that FTIR spectroelectrochemistry can be used as a technique for dynamic and structural studies of redox proteins under near-native conditions in aqueous solution.