Detergent-solubilized dimeric and monomeric cytochrome oxidase (CcO) have significantly different quaternary stability when exposed to 2?3 kbar of hydrostatic pressure. first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the producing 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO made up of all 13 subunits. However, dimeric CcO, in which the association of VIa buy 1001913-13-8 and VIb is usually 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 buy 1001913-13-8 dimeric form of cytochrome oxidase, i.e., stabilization of its quaternary structure. Bovine heart cytochrome oxidase (EC 1.9.3.1, CcO)1 is the terminal complex of the mitochondrial respiratory chain. It is a multisubunit proteinCphospholipid complex consisting of 13 dissimilar subunits, three or four tightly bound cardiolipins, and four metal centers (CuA, heme oxidase, but not within the dimeric enzyme. These results suggest that dimerization may be essential for maintaining the maximum structural stability of this multisubunit, integral membrane protein complex. EXPERIMENTAL PROCEDURES Materials. Dodecyl maltoside was purchased from Anatrace Inc. Sodium cholate and horse heart cytochrome (type III) were purchased from Sigma Chemical Co. The C18 reversed-phase HPLC column (4.6 mm 250 mm, 218TP104, 5 as buy 1001913-13-8 the substrate. Preparation of Dimeric, Monomeric, and 11-Subunit Monomeric Cytochrome c Oxidase. CcO dimer, made up of two copies of each of the 13 CcO subunits, was prepared by solubilizing 10 oxidized per mole of CcO per second) was measured spectrophotometically by following the pseudo-first-order rate of ferrocytochrome oxidation. Enzyme assay conditions were as follows: 1.75 nM buy 1001913-13-8 CcO, 25?30 oxidase. The electron transfer activity of dimeric (), 13-subunit monomeric (), and 11-subunit monomeric CcO () was DLL4 measured as a function of exposure time to 3 kbar … Hydrostatic Pressure-Induced Perturbation of CcO Visible and Fluorescence Spectra. With our gear, absorbance spectra cannot be collected in real time during exposure of CcO to high hydrostatic pressure. Absorbance spectra can only be obtained after decompression and removal of the sample from your pressure cell. Using this approach, no changes are detected in the visible spectrum of monomeric or dimeric CcO after exposure to 3 kbar of pressure for 2 h. Therefore, perturbation of the heme environment is usually unlikely to buy 1001913-13-8 be responsible for the pressure-induced inactivation of CcO. Real-time fluorescence spectra can be collected during hydrostatic compression. A small reversible change is usually detected in the tryptophan fluorescence spectrum of each type of CcO, but this switch does not correlate with CcO inactivation. The fluorescence switch that occurs with monomeric or dimeric CcO is nearly identical. In each case, exposure to elevated hydrostatic pressure produces an 2 nm reddish shift in the tryptophan emission maximum (emmax increases from 328 to 330 nm) with a concomitant 20?25% decrease in the maximum fluorescence intensity, suggesting a slightly increased level of solvent exposure and a decreased fluorescence lifetime for at least some of the 55 tryptophans within CcO. The tryptophan fluorescence spectrum nearly earnings to normal immediately upon decompression, with dimeric and monomeric CcO having 96 and 90% of their initial fluorescence intensity, respectively. Sedimentation Velocity Analysis of CcO after Exposure to Elevated Hydrostatic Pressure. The distribution of sedimentation coefficients (Oxidasea Conversation Elevated hydrostatic pressure was successfully utilized to probe the functional and structural stability of various oligomeric forms of bovine heart CcO. Dimeric CcO is usually highly resistant to increased hydrostatic pressure, while monomeric CcO is not. Dimerization must either strengthen subunit interactions within the detergent-solubilized complex or lock the complex into a highly resistant structure. Pressure-induced inactivation is not readily reversible, and the structural perturbations persist hours after decompression. The incomplete recovery of tryptophan fluorescence, the increased CcO (24), suggesting that dissociation of subunit VIIa is responsible for the pressure-induced activity loss. Alternatively, the loss in activity may be due to a pressure-induced structural perturbation in CcO, which indirectly results in the dissociation of both subunits. At present, it is not possible to differentiate between the two possibilities. The subunit VIIa-linked mechanism is usually intriguing since we previously found that dissociation of subunit VIIa correlates with peroxide-induced inactivation of CcO (25). Furthermore, subunit VIIa is usually involved in the binding.