Phospholipids in Subunit III Regulate Structural Rearrangements in Cytochrome c Oxidase of Rhodobacter sphaeroides

Abstract

Purified cytochrome c oxidase (COX) from Rhodobacter sphaeroides contains 5 phospholipids per mole as measured using inductively coupled plasma-mass spectrometry. Two phospholipids in the 3-D structure are located within subunit III (SUIII) and four at the interface between SU I, III and IV. Phospholipase A2 treatment of COX for 3 hours at 4 °C in 20 mM MOPS pH 7.2, 20 mM CaCl2 and 0.2 % dodecyl maltoside removes phospholipid (< 1 mole phospholipid per mole enzyme) from COX, results in a 30% decrease in electron transfer activity, and induces suicide inactivation. Both effects on activity were reversed by the addition of 1 mg/ml asolectin, 0.5 mM cardiolipin [CL](with C18:1 side chains) or 0.5 mM fatty acids (>12 carbons) including arachadonic acid. Other common membrane phospholipids (all with C18:1 side chains) had no effect. CL exhibited two sites of interaction with the delipidated enzyme, a high (Km = 0.14 μM) and low affinity (Km = 26 μM) site. Fluorescence anisotropy measurements with enzyme labeled with AEDANS specifically in SUIII exhibited a 50% increase in the rotational rate upon lipid depletion, indicating increased flexibility of SU III within the enzyme's tertiary structure. Additionally, Förster resonance energy transfer between AEDANS and fluorescently labeled CL bound in the high affinity CL binding site (located in the v-cleft of SU III) of the delipidated enzyme indicated that movement towards the active site in SU I occurs upon a change in the redox state of the enzyme. In conclusion, these results show that the endogenous phospholipids regulate enzyme conformation during redox activity by maintaining the structural integrity of the enzyme at the active site.