Labeling of bovine heart cytochrome c oxidase with analogs of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

Abstract

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.

Methylenecyclopropane Rearrangement as a Probe for Free Radical Substituent Effects. σ • Values for Potent Radical-Stabilizing Nitrogen-Containing Substituents

“…Attempts to formylate phenylazo- tert -butane − using POCl 3 /DMF or SnCl 4 /CHCl 2 OMe 29 led to recovery of starting material, but the following route gave the requisite azoaldehyde 25 in low yield. p- Formylbenzoic acid was converted 30 to p- formylphenylisocyanate 23 , which was then reacted with tert -butylamine to form Schiff base urea 24 . Hypochlorite oxidation 33 and hydrolysis afforded p -formylphenylazo- tert -butane 25 , the precursor to 10 , while treatment of 25 with m -chloroperbenzoic acid 34 gave azoxy compound 26 , the precursor to 11 .…”

Photolabeling of Cardiolipin Binding Subunits within Bovine Heart CytochromecOxidase

“…Previous attempts to locate the CL binding sites within CcO using chemical labeling approaches have led to relatively vague subunit assignments. CL has been reported to bind not only to one of the larger subunits, that is, I-IV (16), but also to one or more of the smaller subunits, that is, IV-VIII, (5,(17)(18)(19). The major difficulty encountered in each of these previous studies was that CL-modification of a CcO subunit(s) perturbed its migration on SDS-PAGE making positive identification impossible.…”

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

“…Whether or not CL has a functional role, CL is known to specifically interact with cytochrome c oxidase. Spin-labeled derivatives of CL preferentially bind to the CL-depleted enzyme (Cable & Powell, 1980;Knowles et al, 1981;Powell et al, 1985Powell et al, , 1987, and chemically reactive derivatives of CL preferentially react with some of the cytochrome c oxidase subunits (Kuppe et al, 1987;Dale & Robinson, 1988b;Fowler et al, 1988). The reason for the inconsistencies regarding the functional role of CL is not clear, but the widely different experimental approaches and the different sources of enzyme, e.g., yeast, dogfish, bovine liver, and bovine heart, as well as low estimates of CL content, may explain the different interpretations.…”