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 wild-type and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine, and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wild-type. The detergent inhibition is overcome in the presence of micromolar concentrations of steroids and porphyrin analogues 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 wild-type 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 a(3). 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.

Figure 1

Stimulation of E101A RsCcO by amphipathic carboxylates in 0.06% DDM. Purified E101A RsCcO was assayed in 0.06% DDM as described in Materials and Methods. Panel A: bilirubin (BR; red), cholate (purple), cholesteryl hemisuccinate (CHS; green), deoxycholate (DOC; black), protoporphyrin IX (PPIX; magenta). Panel B: arachidonic acid (cyan), docosahexanoic acid (blue), phytanic acid (orange), retinoic acid (brown). Note different concentration and activity axes in panels A and B; higher affinity ligands are shown in panel B. The starting activities in the absence of ligand ranged from 31–66 s⁻¹.

Figure 2

Effects of DDM (A) and amphipathic carboxylates in low DDM (B). Purified E101A (circles) and WT (triangles) RsCcO were assayed in 0.01% DDM as described in Materials and Methods. Additive in panel A: DDM; additives in panel B: cholesteryl hemisuccinate (CHS; open circles, dashed line), deoxycholate (DOC; solid circles, solid line), retinoic acid (half filled circles, dotted line).

Figure 3

Inhibition of RsCcO by lysolipids and detergents. Purified E101A (A,C) and WT (B) RsCcO were assayed in 0.01% DDM (A,B) and 0.06% DDM (C) as described in Materials and Methods. Additives: CHOBIMALT (red), C12E8 (purple), lyso-PC (black), Triton X100 (blue), Tween 20 (green).

Figure 4

Competition of Triton X100 (A) and CHOBIMALT (B) with deoxycholate (DOC) for activation of E101A RsCcO. Purified E101A RsCcO was assayed in 0.01% DDM as described in Materials and Methods in the absence (solid circles) and presence (open circles) of 300 μM DOC.

Figure 5

Spectra of purified E101A and WT CcO with and without CHOBIMALT. Panel A: Soret region; panel B: α region. Spectra were normalized to the fully reduced α peak height at 607nm – 630nm. Spectra of fully oxidized and fully reduced purified CcO are shown in black. Steady-state spectra in 100mM CHES pH 9.5 with 0.01% DDM were recorded 3 minutes after adding 1 mM ascorbate and 200 μM TMPD: WT only (green), WT with CHOBIMALT (blue), E101A only (magenta), E101A with CHOBIMALT (red).

Figure 6

Potential binding orientations of known amphipathic ligands in the RsCcO steroid binding site. (A) Heme (magenta sticks) and cholate (black sticks) bound in the same site in two different crystal structures of ferrochelatase (PDB: 2PO7 and 2QD2). (B) An energetically favorable orientation of the BR analog biliverdin (red sticks) in RsCcO based on ligand transposition from serum albumin. Energetically favorable SLIDE dockings into RsCcO of (C) PPIX (magenta sticks), (D) BR (red sticks), and (E) two DDM flexible conformers (yellow and magenta sticks). (B–E) The crystallographically bound deoxycholate (black sticks; PDB: 3DTU) and DDM (blue sticks; PDB 3HB3) are shown as reference molecules.

Figure 7

RsCcO deoxycholate binding site with conserved residues and bound water molecules (PDB 3DTU). The steroid binding site side chains are colored by degree of conservation in CcO sequences as calculated in Buhrow et al. where residues with >75%, 50–75%, or less than 50% conservation are colored dark blue, medium blue, and gray, respectively. The structure includes a bound deoxycholate (green sticks), K-path water molecules (red spheres), and a cadmium ion (orange sphere).

Figure 8

RsCcO K-path rigidification upon ligand binding. (A) RsCcO (PDB: 2GSM) and (B) deoxycholate-bound RsCcO (PDB: 3DTU) are colored by crystallographic B-value, where red represents values ≥ 65 Ų, while deep blue represents values ≤ 35 Ų. (C) RsCcO and (D) deoxycholate-bound RsCcO are colored by internal protein flexibility as determined by the ProFlex. Deep blue represents greatest rigidity while red represents greatest flexibility.

Figure 9

Two site model for binding of steroids and detergents near the K-path entrance. The lower site is shown with the crystallographic deoxycholate in yellow (from RsCcO, PDB: 3DTU). The upper site is shown with the crystallographic DDM in green (from PdCcO, PDB: 3HB3) modeled with the flexible head group in the most extended position. The surface of the RsCcO structure is represented with the hydrophobic regions in dark orange, the hydrophilic charged regions in blue, and neutral regions in pale blue/pale orange/white (Chimera, UCSF). The position of the membrane region is indicated by blue lines.