Modulation of the Conformation of Cytochrome c Oxidase from paracoccus Denitrificans by Active-Site Mutations

We have measured the resonance Raman spectra of the wild-type (wt) and 8 different mutants of cytochrome c oxidase from Paracoccus denitrificans (pdCcO). Most of the mutants bring about large changes in the binuclear center including conversion of heme a3 from high spin to low spin and/or disruption of the H-bonding environments surrounding the formyl groups of both heme a and heme a3. To obtain a semi-quantitative measure of the conformational changes induced by the mutations, we use CO as a structural probe. In CcO, the Fe-CO moiety typically exhibits two conformations, called the α and β forms. The Fe-CO stretching mode of the α form is present at ∼ 520 cm−1, whereas that of the β forms appears at ∼490-495 cm−1. The α form, which is the active conformation of the enzyme, has Fe-CO and C-O stretching modes that do not fall on the νFe-COvs νC-O inverse correlation line characteristic of heme coordinated by a histidine ligand, presumably owing to the interaction of the CO with the nearby CuB atom in the binuclear center. Our data of the CO-bound pdCcO showed that α/(α+β) intensity ratio varies from nearly zero to one in the mutants. The changes in the α/(α+β) ratio correlate well with changes in some of the heme modes. We postulate that the conformation of the catalytic site, consisting of the two heme groups and CuB, is perturbed by the mutations, as indicated by the changes in the heme modes, which disrupts of the juxtaposition between CuB and the iron atom of heme a3 as reflected by the changes in the α/(α+β) ratio. The implications of these results in relation to the measured functional properties of the enzyme will be discussed.