Investigating the Many Roles of Internal Water in Cytochrome c Oxidase

Abstract

Cytochrome c oxidase (C cO) is the terminal enzyme in the respiratory electron transport chain. As part of its catalytic cycle, C cO transfers protons to its Fe-Cu binuclear center (BNC) to reduce oxygen, and in addition, it pumps protons across the mitochondrial inner, or bacterial, membrane where it is located. It is believed that this proton transport is facilitated by a network of water chains inside the enzyme. Here we present an analysis of the hydration of C cO, including the BNC region, using a semi-empirical hydration program, Dowser++, recently developed in our group. Using high-resolution X-ray data, we show that Dowser++ predictions match very accurately the water molecules seen in the D- and K-channels of C cO, as well as in the vicinity of its BNC. Moreover, Dowser++ predicts many more internal water molecules than is typically seen in the experiment. However, no significant hydration of the catalytic cavity in C cO described recently in the literature is observed. As Dowser++ itself does not account for structural changes of the protein, this result supports the earlier assessment that the proposed wetting transition in the catalytic cavity can only either be due to structural rearrangements of BNC, possibly induced by the charges during the catalytic cycle, or occur transiently, in concert with the proton transfer. Molecular dynamics simulations were performed to investigate the global dynamic nature of Dowser++ waters in C cO, and the results suggest a consistent explanation as to why some predicted water molecules would be missing in the experimental structures. Furthermore, in light of the significant protein hydration predicted by Dowser++, the dielectric constant of the hydrated cavities in C cO was also investigated using the Fröhlich-Kirkwood model; the results indicate that in the cavities where water is packed sufficiently densely the dielectric constant can approach values comparable even to that of bulk water.

Conformational Motions and Water Networks at the α/β Interface in E. coli Ribonucleotide Reductase

“…Thus, the MD simulations suggest that the proton acceptor for Y356 is a water molecule in the interfacial region, and presumably this proton is subsequently shuttled to bulk solvent. Water networks allowing redox active enzymes to shuttle protons to solvent have also been characterized computationally in other proteins. , …”

“…Water networks allowing redox active enzymes to shuttle protons to solvent have also been characterized computationally in other proteins. 57,58 Previously, Y356 and Y731 were proposed to communicate across the interface on the basis of EPR experiments indicating that the local environment around Y356 is influenced by Y731 via mutation studies. 56 However, a direct hydrogen bond between these two residues is not supported by H 1 ENDOR data 44 or the distances in the cryo-EM structure.…”

Interplay of Hydration and Protonation Dynamics in the K-Channel of Cytochrome c Oxidase

“…Getting the proton to K362 has been considered as an important step/pre-requisite for proton transport through the K-channel [ 9 , 13 , 37 ]. While a number of previous simulation studies suggest a water-mediated hydrogen-bonded network between K362 and Y288 [ 17 , 18 , 20 , 38 ], partially via T359, regardless of K362’s protonation state, with neutral K362 no connectivity with the lower part of the K-channel has been observed [ 18 , 38 ] but with a protonated K362 there is enough water in the lower part to render water chains between K362 and E101 conceivable [ 20 ]. Our simulation data agree in as much as protonated K362 leads to a high connectivity of hydrogen bonds in the upper and lower part of the channel.…”

“…Hydration levels in the cavities of the BNC and in the proton-conducting channels in CcO have been studied by prediction of internal water sites and molecular dynamics simulations [ 17 , 18 , 19 ]. The formation of water wires or water chains, along which a proton transport can also take place in the channels [ 6 , 14 , 20 ] or from the BNC to the P-side and thus the exterior of the protein has been probed by simulations [ 21 ].…”

Protein Motifs for Proton Transfers That Build the Transmembrane Proton Gradient

“…Although we refer to “protons”, H + does not travel alone. Rather it is associated with a water (hydronium, H 3 O + ) or two water molecules as a Zundel cation (H 5 O 2 + ) or as a larger, Eigen complex (H 9 O 4 + ) ( Agmon, 1995 ; Wraight, 2006 ; Farahvash and Stuchebrukhov, 2018 ). In proteins, the proton can also be bound to redox cofactors, to acidic or basic residues or trapped as a stabilized hydronium ( Xu and Voth, 2006 ; Freier et al, 2011 ; Ikeda et al, 2017 ).…”