Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

Abstract

Cytochrome c oxidase (CytcO) is a redox-driven, membranebound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogenbonded water molecules that span the distance between an aspartate (Asp 132 ), near the protein surface, and glutamate Glu 286 , which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu 286 affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser 197 (S197A or S197D), located ϳ7 Å from Glu 286 . Although Ser 197 is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of ϳ35% of that of the wild-type CytcO, and the O 2 reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

Functional Hydration and Conformational Gating of Proton Uptake in Cytochrome c Oxidase

“…2b), together with the multimodal character of the spatial water density distribution and its tolerance to changes in hydration number, are consistent with the relative tolerance of the enzyme to mutations in this region of the D-channel. 25,44,64 In addition, these properties have several implications to the mechanism of proton uptake, both in the wild type and in N139 mutants.…”

A D-Pathway Mutation Decouples the Paracoccus denitrificans Cytochrome c Oxidase by Altering the Side-Chain Orientation of a Distant Conserved Glutamate

“…1), have been identified by analyzing both structural data [3][4][5][6][7][8] and mutagenesis studies. [9][10][11][12][13][14][15][16][17][18][19][20] These pathways connect the inner aqueous phase and the binuclear site. The initial concept that protons consumed in water formation at the active site use the K-pathway and translocated protons are transferred via the Dpathway 3 could not be maintained because measurements of charge translocation 21,22 and results from flow-flash experiments 23,24 indicated that the K-pathway delivers the first one or two protons to the binuclear center during the reductive phase of the catalytic cycle only, with the remaining two or three substrate protons transported via the D-pathway during the "oxidative" part of the cycle (after the binding of molecular oxygen to the reduced binuclear center of COX).…”

Proton-coupled gating in chloride channels

“…However, there is another glutamate adjacent to Glu in (E202 in figure 1) that is conserved in the mammalian CLCs and which could possibly substitute as a titratable residue along the proton permeation pathway. Indeed, malleability of proton pathways has been observed with other proton-transport proteins including cytochrome c oxidase ( Namslauer et al 2007) and bacteriorhodopsin (Sonar et al 1994).…”