Broken Symmetry DFT Calculations/Analysis for Oxidizedand Reduced Dinuclear Center in Cytochrome c Oxidase:Relating Structures Protonation States Energies and MössbauerProperties in ba3Thermus thermophilus

摘要

The Fea3³⁺···CuB²⁺ dinuclear center (DNC) structure of the as-isolated oxidized ba3 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 ba3 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 Fea3²⁺···CuB⁺ DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779–5783) revealed multiple ⁵⁷Fea3²⁺ and ⁵⁷Fea3³⁺ components. Moreover, one of the ⁵⁷Fea3³⁺ 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 ⁵⁷Fea3³⁺ component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauerspectra and X-ray crystal structures, the spin crossover of one ofthe ⁵⁷Fea3³⁺ components, and howthe coordination and spin states of the Fea3^3+/2+ and Cu^2+/1+ sites relate to the heterogeneity of theDNC structures, we have applied density functional OLYP calculationsto the DNC clusters established based on the different X-ray crystalstructures of ba3 CcOfrom Tt. As a result, specific oxidized and reducedDNC structures related to the observed Mössbauer spectra andto spectral changes with temperature have been proposed. Our calculationsalso show that, in certain intermediate states, the His233 and His283ligand side chains may dissociate from the CuB⁺ site, and they may become potential proton loading sites duringthe catalytic cycle.