A Conserved Amphipathic Ligand Binding Region Influences K-Path-Dependent Activity of Cytochrome c Oxidase

Abstract

A conserved, crystallographically-defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO, as well as altering the activity of wildtype and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wildtype. The detergent inhibition is overcome in the presence of μM concentrations of steroids and porphyrin analogs including deoxycholate, cholesteryl hemisuccinate, bilirubin, and protoporphyrin IX. In addition to alleviating detergent inhibition, amphipathic carboxylates including arachidonic, docosahexanoic, and phytanic acids stimulate the activity of E101A to wildtype levels by providing the missing carboxyl group. Computational modeling of dodecyl maltoside, bilirubin, and protoporphyrin IX into the conserved steroid site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunit I and II, including the entrance to the K-path and helix VIII of subunit I, mediates the observed competitive ligand interactions involving two overlapping sites. Spectral analysis indicates that ligand binding to this region affects CcO activity by altering the K path dependent electron transfer equilibrium between heme a and heme a3. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance.

Functions of the hydrophilic channels in protonmotive cytochrome c oxidase

“…rsif.royalsocietypublishing.org J R Soc Interface 10: 20130183 had no effect [53] suggesting instead that the entry route may be formed by a more extended region that does not obligatorily involve the E62 II residue [49]. This notion has gained support from a recent study that further suggests that this region can bind a range of compounds that may modulate K channel behaviour [54].…”

The K-path entrance in cytochrome c oxidase is defined by mutation of E101 and controlled by an adjacent ligand binding domain

“…In one of the crystal structures of PdCcO [PDB: 3HB3] this site is occupied by the alkyl tail of an unusual, upside-down DDM molecule whose head group overlaps with the steroid site [29]. It was hypothesized that if the tails of long-chain ligands bind in this upper site, their head groups could compete with or displace steroid-like ligands that bind in the lower site, consistent with observed ligand effects and with modeling of the positioning of the DDM head group [21]. …”

“…Although most of the compounds that stimulated E101A were amphipathic carboxylates, several non-ionic detergents also interacted competitively with the site and modified the effects of other ligands [21]. In particular, the detergent dodecyl maltoside (DDM) strongly inhibited E101A activity, in contrast to its support of maximum activity of WT RsCcO [5] and bovCcO [22, 23].…”

“…In particular, the detergent dodecyl maltoside (DDM) strongly inhibited E101A activity, in contrast to its support of maximum activity of WT RsCcO [5] and bovCcO [22, 23]. Different activity levels observed for K-path mutants of RsCcO [15, 18, 19, 21] and PdCcO [16] may be explained by use of different DDM levels in assays. In contrast, Tween20 is a strong activator of RsCcO [21] and bovCcO [24–26].…”

“…To account for observed ligand interactions, a two-site model [21] was postulated involving “lower” and “upper” sites. The lower site was defined as the crystallographically-observed binding site near E101 for DOC in RsCcO and cholate in bovCcO.…”