FTIR Detection of Protonation/Deprotonation of Key Carboxyl Side Chains Caused by Redox Change of the CuA-HemeaMoiety and Ligand Dissociation from the Hemea3-CuBCenter of Bovine Heart CytochromecOxidase

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Abstract
FTIR spectral changes of bovine cytochrome c oxidase (CcO) upon ligand dissociation from heme a3 and redox change of the CuA-heme a moiety (CuAFea) were investigated. In a photosteady state under CW laser illumination at 590 nm to carbonmonoxy CcO (CcO−CO), the C−O stretching bands due to Fea32+CO and CuB1+CO were identified at 1963 and 2063 cm-1, respectively, for the fully reduced (FR) state [(CuAFea)3+Fea32+CuB1+] and at 1965 and 2061 cm-1 for the mixed valence (MV) state [(CuAFea)5+Fea32+CuB1+] in H2O as well as in D2O. For the MV state, however, another band due to CuB1+CO was found at 2040 cm-1, which was distinct from the α/β conformers in the spectral behaviors, and therefore was assigned to the (CuAFea)4+Fea33+CuB1+CO generated by back electron transfer. The FR-minus-oxidized difference spectrum in the carboxyl stretching region provided two negative bands at 1749 and 1737 cm-1 in H2O, which were apparently merged into a single band with a band center at 1741 cm-1 in D2O. Comparison of these spectra with those of bacterial enzymes suggests that the 1749 and 1737 cm-1 bands are due to COOH groups of Glu242 and Asp51, respectively. A similar difference spectrum of the carboxyl stretching region was also obtained between (CuAFea)3+Fea32+CuB1+CO and (CuAFea)5+Fea32+CuB1+CO. The results indicate that an oxidation state of the (CuAFea) moiety determines the carboxyl stretching spectra. On the other hand, CO-dissociated minus CO-bound difference spectra in the FR state gave rise to a positive and a negative peaks at 1749 and 1741 cm-1, respectively, in H2O, but mainly a negative peak at 1735 cm-1 in D2O. It was confirmed that the absence of a positive peak is not caused by slow deuteration of protein. The corresponding difference spectrum in the MV state showed a significantly weaker positive peak at 1749 cm-1 and an intense negative peak at 1741 cm-1 (1737 cm-1 in D2O). The spectral difference between the FR and MV states is explained satisfactorily by the spectral change induced by the electron back flow upon CO dissociation as described above. Thus, the changes of carboxyl stretching bands induced both by oxidation of (CuAFea) and dissociation of CO appear at similar frequencies (∼1749 cm-1) but are ascribed to different carboxyl side chains.

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