Abstract
Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.
Highlights
Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples O2 reduction to proton pumping
The unit-cell parameters were very similar to those of the oxidized crystal, with deviations of $0.1%, which are comparable to those for crystals obtained at pH 5.7 (Yano et al, 2016). This observation indicates that the CcO molecules in the oxidized and the reduced crystals were very similar in terms of their structures, which affect the molecular packing within the crystals
The structural alterations detected at pH 5.7 to Asp51, the magnesium-containing water cluster, the haems and helix X upon redox change, which provide important structural evidence for the H-pathway proton-pumping mechanism, were reproduced at pH 7.3
Summary
Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples O2 reduction to proton pumping. Mammalian CcO is located in the inner mitochondrial membrane and accepts electrons from cytochrome c in the intermembrane space (P-side). The protons used for H2O synthesis are supplied to the enzyme via the D-pathway and K-pathway from the matrix side (N-side), whereas pumping protons are transferred through the enzyme from the N-side to the intermembrane space (P-side) in order to generate a proton-concentration gradient across the mitochondrial inner membrane (Yoshikawa & Shimada, 2015). We reported the structures of four redox-active metal sites [haem a (Fea), haem a3 (Fea3), CuB and CuA; Tsukihara et al, 1995] and the whole-protein structure of the oxidized state (Tsukihara et al, 1996), and noted the structural differences between the oxidized and reduced CcO crystals (Yoshikawa et al, 1998). The proton-pumping mechanism, later named the H-pathway mechanism, was proposed based on a structural alteration in
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