Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Abstract

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm −1 range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(−), and 3,607(+) cm −1 . A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D 2 O and H 2 18 O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.energy coupling | mitochondria | respiratory chain | complex IV M itochondrial cytochrome c oxidase (CcO) catalyses electron transfer from cytochrome c to molecular oxygen, conserving the released energy as coupled transmembrane proton transfers. It is a member of a homologous "superfamily" of oxidases that includes diverse bacterial forms; all types in the CcO subgroup contain a common "catalytic core" of subunits I, II, and III with equivalent metal centers of Cu A , heme a. and a binuclear center (BNC) that is formed from heme a 3 and Cu B (1, 2). Structures of bovine mitochondrial (3, 4) and various bacterial (5-7) CcOs have been solved at atomic resolution and show common details in the binuclear heme a 3 ∕Cu B catalytic site, other cofactors, key amino acids, and several unusual structural features. These structural data, coupled with a substantial body of biophysical and spectroscopic studies, have provided detailed information on internal electron transfer and the chemical catalytic cycle of oxygen reduction.Crucially, however, these structural data have not provided a single picture for all forms of CcO of the pathway(s) for coupled proton transfer and, therefore, the interplay between electron transfer/oxygen chemistry and the intraprotein proton transfer reactions that result in proton-motive action. Crystal structures of bacteri...

Insights into the Mechanism of Proton Transport in Cytochrome c Oxidase

“…The recent FTIR experiment also confirmed that the hydrogen bond network rearrangement occurs in response to the oxidation state change. 34 Also, Wikström, et al reported that the hydrogen bond network of the water cluster in the non-polar cavity is changed by change of oxidation states of heme a and the BNC, although their empirical MD simulations included no excess proton. 13 …”

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…31 Later, dangling and weakly H-bonded water molecules have been also detected at room temperature. [32][33][34] Changes in the D-O-D bending of water molecules have been detected, as well. 35 It was shown that the exchange of the protein solvent from H 2 16 O to H 2 18 O is enough to detect dangling waters in the dark state of BR, without the need to induce a photoreaction.…”

Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase

“…1A, and see below). Even though there is little direct experimental support available for such a water-gated mechanism, a recent FTIR study indeed suggests changes in water organization upon changes in the redox state of the enzyme (11). Many of the elementary steps that were postulated in the water-gated mechanism have gained support from experiments in the recent past (12,13).…”