The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O-2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O-2 with the mixed valence form (Cu-A(2+), heme a(3+), heme a(3)(2+)-Cu-B(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (P-M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P-M intermediate is also formed in the reaction of Y167F with O-2. These results suggest that in the fully reduced enzyme, the proton pumping nu(Fe(IV)=O) = 804 cm(-1) to nu(Fe(IV)=O) = 790 cm(-1) transition (P -> F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a(3) but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a(3). The implications of these results with respect to the role of an O=Fe(IV)His-411-H-bonded form to the ring A propionate of heme a(3)-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P -> F transition are discussed. We propose that the environment proximal to the heme a(3) controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.