Differential Stability of Dimeric and Monomeric Cytochrome c Oxidase Exposed to Elevated Hydrostatic Pressure

Abstract

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2−3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time-and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, highperformance ion-exchange chromatography, and reversed-phase and SDS-PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure.Bovine heart cytochrome c oxidase (EC 1.9.3.1, CcO) 1 is the terminal complex of the mitochondrial respiratory chain. It is a multisubunit protein-phospholipid complex consisting of 13 dissimilar subunits, three or four tightly bound cardiolipins, and four metal centers (Cu A , heme a, heme a 3 , and Cu B ) with a combined molecular weight of ∼205000 for the monomeric enzyme (1-3). CcO crystallizes as a dimer, which is generally seen as the functional unit within the inner membrane; however, little or no information is available regarding the functional or structural significance of a dimeric structure. For example, monomeric and dimeric CcO are both highly active in terms of electron transfer (4,5). The dimeric form of † This work was supported by grants from the National Institutes of Health (GMS 24795) and The Robert A. Welch Foundation (AQ1481).* To whom correspondence should be addressed. Telephone: (210) 567−3754. Fax: (210) 567−6595. E-mail: robinson@uthscsa.edu. ‡ Present add...

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