The propionate groups of heme a and a3 in cytochrome c oxidase (CcO), have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a3, as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H2O-D2O isotope substitution experiments, combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).

Pyridine generally acts as the proton acceptors in the hydrogen bonding interaction by using its lone pair n(N) or pi-electrons. Some previous research indicated that for the N-type H-bond, the ring breathing mode v(1), the N-para-C stretching mode v(6a) and the meta-CC stretching mode v(8a) of pyridine showed a frequency blueshift but the triangle mode v(12) had no change in frequency. Both electrostatic interaction and charge transfer caused by intermolecular hyperconjugation n(N)-->sigma( *)(HX) have contributions to the frequency blue shifts, while charge transfer is predominant at equilibrium intermolecular distance. An intramolecular hyperconjugation between the lone pair n(N) and the two sigma( *)(meta-CC) orbitals in the pyridine ring provides a reasonable interpretation for the effect of charge transfer on the ring stretching modes upon formation of the N-type H-bonding.

The structural and functional properties of active site mutants of cytochrome c oxidase from Paracoccus denitrificans (PdCcO) were investigated with resonance Raman spectroscopy. Based on the Fe-CO stretching modes and low frequency heme modes, two conformers (α- and β-forms) were identified that are in equilibrium in the enzyme. The α-conformer, which is the dominant species in the wild type enzyme, has a shorter heme a3 iron-CuB distance and a more distorted heme, as compared to the β-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the α-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the α-conformer is the active form of the enzyme. The data suggest that the native structure of the enzyme is in a delicate balance of intramolecular interactions. We present a model in which the mutations destabilize the α-conformer, with respect to the β-conformer, and raise the activation barrier for the inter-conversion between the two conformers. The accessibility of the two conformers in the conformational space of CcO plausibly plays a critical role in coupling the redox reaction to proton translocation during the catalytic cycle of the enzyme.

The redistribution of two electrons in the four redox centers of cytochrome c oxidase following photodissociation of CO from the CO-bound mixed valence species has been examined by resonance Raman spectroscopy. To account for both the kinetic data, obtained from 5 s to 2 ms, and the equilibrium results, a model is proposed in which the electron redistribution is modulated by a protein conformation transition from a nascent P 1 state to a relaxed P 2 state in a time window longer than 2 ms. In this model, all six possible two-electron reduced species are considered. The high population of species with a one-electron reduced binuclear center, in which the spectrum of heme a 3 is perturbed by the redox state of Cu B , accounts for the significant residuals in the fitting of the kinetic data with four standard spectra derived from redox species with either zero or two electrons in the binuclear center. Under equilibrium conditions, the conformational change to the P 2 state destabilizes the redox states with only one electron in the binuclear center with respect to those with either zero or two electrons. As a result, the redox equilibrium is perturbed, and the electrons are redistributed. A simulation based on the new kinetics scheme, in which the electron redistribution is modulated by the protein conformation, gives reasonable agreement with both the equilibrium and the kinetic data, demonstrating the validity of this model.