Cardiolipin-depleted bovine heart cytochrome c oxidase: binding stoichiometry and affinity for cardiolipin derivatives

Abstract

Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-[14C]acetylcardiolipin (AcCL), Kd,app less than 0.1 microM, that are not present in enzyme containing endogenous CL. An additional 2-3 lower affinity AcCL binding sites, Kd,app = 4 microM, are present in the CL-depleted complex, but these sites are also present in enzyme containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) compete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to the high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The presence or absence of hydroxyl groups in CL, MLCL, or DLCL also has little effect upon binding affinities. Binding specificity clearly favors CL since phosphatidylglycerol, phosphatidic acid, and phosphatidylcholine each have very low affinity for the CL binding sites (Kd,app greater than 20 microM). We, therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.

Photolabeling of Cardiolipin Binding Subunits within Bovine Heart CytochromecOxidase

“…Bis-(AzC 12 )-CL photolabeling of monomeric, CL-containing CcO, which contains four filled CL binding sites, slightly decreased the yields of subunits VIIa, VIIb/ VIIc, and VIII ( Figure 5A), but the errors associated with these decreases were nearly as large as the values themselves. The poor labeling of these subunits is almost certainly due to incomplete exchange of bis-(AzC 12 )-CL for two endogenous CL that are tightly bound near these subunits ([ 14 C]-acetyl-CL binds at the two high-affinity sites only if the endogenous CL has been removed [13]). The inability of bis-(AzC 12 )-CL to exchange for endogenous CL would explain why the photolabeling of these subunits is not significantly higher than the nonspecific photolabeling of other transmembrane subunits, that is, subunits IV and VIc.…”

“…No other subunits dissociate from CcO upon removal of these tightly bound CL, but the resulting CL-free 11-subunit complex has only half of its original electron transport activity. Cardiolipin binding studies confirm the presence of two high-affinity and one or two lower-affinity binding sites (13). CcO, therefore, contains two types of CL binding sites: (1) two high-affinity sites that regulate electron transport and (2) one or two lower-affinity sites that stabilize subunits VIa and VIb and, therefore, CcO dimerization.…”

“…The evidence for this second site is as follows. (1) Previous CL binding data indicate that two, not one, CLs bind to 11-subunit CL-free CcO (13). (2) Only subunits VIIa, VIIc, and possibly VIII are photolabeled by either arylazido-CL derivative.…”

Synthesis and Inhibition Mechanism of Δlac-Acetogenins, a Novel Type of Inhibitor of Bovine Heart Mitochondrial Complex I

“…For instance, the presence of bound cardiolipin is essential for full activity of the ADP/ATP carrier (26), cytochrome c oxidase (27), and the cytochrome bc 1 complex (28,29). The high-resolution structures of bound cardiolipin(s) have been described for the cytochrome bc 1 complex (30).…”

Cardiolipin Biosynthesis and Mitochondrial Respiratory Chain Function Are Interdependent

“…Thus, a detailed understanding of the regulation of CL levels will provide important insights into the manifestation of these conditions. CL is associated with major proteins of the mitochondrial respiratory chain including NADH dehydrogenase (complex I) (13), ubiquinol:cytochrome c reductase (complex III) (13)(14)(15)(16)(17), and cytochrome c oxidase (complex IV) (18,19), the ATP synthase (complex V) (20), and the carrier proteins for phosphate (21) and adenine nucleotides (22). CL modulates the catalytic activities of proteins, as seen in the case of the ADP-ATP carrier (5,23,24) and complex IV (25), and/or provides stability as reported for complex III (17) and complex IV (19).…”