Phospholipase A₂ Digestion of Cardiolipin Bound to Bovine Cytochrome c Oxidase Alters Both Activity and Quaternary Structure^†

Abstract

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.

Photolabeling of Cardiolipin Binding Subunits within Bovine Heart CytochromecOxidase

“…CL-free CcO is also missing subunits VIa and VIb because they dissociate upon CL removal (12). Photolabeling differences between the two types of CcO enabled us to identify which CcO subunits are adjacent to the high-and low-affinity CL binding sites.…”

“…(2) Unbound bis-(AzC 12 )-CL or SAND-gly-CL was removed by anionexchange HiTrap Q column chromatography in the dark (12). (3) The resulting azido-CL/CcO complex (4 μM) was photoactivated by a 30 min exposure to 460 nm monochromatic light in a quartz cuvette at 25 °C using a Fluorolog-3 spectrofluorometer (Jobin Yvon-Spex) equipped with a 450 W xenon lamp.…”

“…A number of basic amino acid side chains are located near the N-terminus of subunits VIIa and VIIc, (e.g., Lys 10 , His 21 and Arg 33 of VIIa; His 2 , Lys 9 , and Arg 20 of VIIc), which would facilitate binding of an anionic PL near this location. Binding of CL at this site would explain the photolabeling of both subunit VIIa and subunit VIIc and would also help to explain the 50% decrease in electron transport activity that occurs upon removal of the two tightly bound CLs (12). Binding of CL3 at site VIIa-B would place the polar headgroup of CL near the entrance to the D-channel, possibly serving as a proton antenna to facilitate proton input into the binuclear center [one pK a of CL is ∼7 (32)].…”

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of cardiolipin extracted from detergent-solubilized mitochondrial electron transfer complexes

“…Cytochrome c oxidase was isolated from bovine heart muscle mitochondrial particles as previously described [6,8]. Purified enzyme was solubilized in 20 mM Tris-SO 4 , pH 7.4, buffer, containing 2 mM dodecyl maltoside.…”

“…Purified enzyme was solubilized in 20 mM Tris-SO 4 , pH 7.4, buffer, containing 2 mM dodecyl maltoside. Most of the phospholipids, except for tightly bound cardiolipin and residues of phosphatidylethanolamine, were removed by HiTrapQ ionexchange column chromatography [8]. Bound phospholipids were extracted from the 2.5-5.0 μM dodecyl maltoside-solubilized cytochrome c oxidase using chloroform/methanol/water extraction [9].…”