Modulation of the active site conformation by site-directed mutagenesis in cytochrome c oxidase from Paracoccus denitrificans.

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 (alpha- and beta-forms) were identified that are in equilibrium in the enzyme. The alpha-conformer, which is the dominant species in the wild-type enzyme, has a shorter heme a(3) iron-Cu(B) distance and a more distorted heme, as compared to the beta-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the alpha-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the alpha-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 alpha-conformer, with respect to the beta-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.