Broken Symmetry DFT Calculations/Analysis for Oxidized and Reduced Dinuclear Center in Cytochrome c Oxidase: Relating Structures, Protonation States, Energies, and Mössbauer Properties in ba(3)Thermus thermophilus

[Image: see text] The Fe(a3)(3+)···Cu(B)(2+) dinuclear center (DNC) structure of the as-isolated oxidized ba(3) cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba(3) CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO’s were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fe(a3)(2+)···Cu(B)(+) DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba(3) CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779–5783) revealed multiple (57)Fe(a3)(2+) and (57)Fe(a3)(3+) components. Moreover, one of the (57)Fe(a3)(3+) components observed at 4.2 K transformed from a proposed “low-spin” state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin (57)Fe(a3)(3+) component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the (57)Fe(a3)(3+) components, and how the coordination and spin states of the Fe(a3)(3+/2+) and Cu(2+/1+) sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba(3) CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the Cu(B)(+) site, and they may become potential proton loading sites during the catalytic cycle.