The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

Abstract

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redoxdriven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 3 Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8͞1.9-Å resolution in the fully oxidized͞ reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redoxcoupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

X-ray structure of cyanide-bound bovine heart cytochromecoxidase in the fully oxidized state at 2.0 Å resolution

“…Comparison of the structures of the oxidized and reduced enzymes revealed significant conformational differences in residues 49-55 of subunit I (Yoshikawa et al, 1998), in residues 380-386 of helix X (371-400) and in the hydroxyfarnesylethyl group of haem a (Tsukihara et al, 2003). Structures showing the redox-coupled changes were compared with those of the CN À -bound oxidized enzyme, as shown in Fig.…”

Dielectric relaxation of cytochrome c oxidase: Comparison of the microscopic and continuum models

“…With Amber atomic radii, a satisfactory cavity size and convergence for all three examined structures ͑the protonated crystal structure 43 and the initial and final state average structures͒ is only achieved using probe radius as small as 1.25 Å. The same optimal value for the parameter was obtained in morphological study of cavities in globular proteins.…”

“…In the linear approximation, any should result in the same reaction-field energy. The continuum PB calculations are typically carried out using the crystal structure of the protein, 43 disregarding the fact that equilibrium protein structures can be different in protonated and deprotonated states. To examine the difference of using different structures, we performed PB calculations for three protein structures: r cs , ͗r͘ 0 , and ͗r͘ 1 , where r cs denotes the crystal structure 43 with optimized hydrogen positions, and ͗r͘ 0 and ͗r͘ 1 denote average configurations of the initial ͑pro-tonated His291͒ and final ͑deprotonated His291͒ equilibrium states of the protein.…”

“…57. The dry CcO is modeled by only subunits A and B taken from the fully reduced bovine heart cytochrome c oxidase structure ͑Tsukihara et al, 43 PDB code 1V55͒ and initially protonated according to the standard protonation state of the residues. 58 The partial charges of the redox centers, heme a, heme a3, Cu A , and Cu B , were obtained by fitting the electrostatic potential in a Hartree-Fock calculation with the 6-31G * basis set.…”

“…The structure and topology of the CcO with internal water were obtained exactly in the same way as for the dry CcO with only the difference that 42 water molecules were added to the system. Residue numbers of the oxygen atoms forming the internal water, according to the crystal structure order, 43 are as follows : 10, 15, 16, 17, 19, 20, 22, 23, 24, 31, 32, 33, 34, 35, 38, 40, 41, 42, 44, 46, 48, 54, 55, 63, 111, 165, 179, 220, 221, 228, 258, 326, 369, 371, 642, 683, 684, 702, 716, 2004, 2288, and 2574. The internal water, in fact, does not correspond to a single continuous cavity.…”