Sequential dissociation of subunits from bovine heart cytochrome C oxidase by urea

Abstract

The quaternary stability of purified, detergent-solubilized, cytochrome c oxidase (CcO) was probed using two chemical denaturants, urea and guanidinium chloride (GdmCl). Each chaotrope induces dissociation of five subunits in a concentration-dependent manner. These five subunits are not scattered over the surface of CcO but are clustered together in close contact at the dimer interface. Increasing the concentration of urea selectively dissociates subunits from CcO in the following order: VIa and VIb, followed by III and VIIa, and finally Vb. After incubation in urea for 10 min at room temperature, the sigmoidal dissociation transitions were centered at 3.7, 4.6, and 7.0 M urea, respectively. The secondary structure of CcO was only minimally perturbed, indicating that urea causes disruption of subunit interactions without urea-induced conformational changes. Incubation of CcO in urea for 120 min produced similar results but shifted the sigmoidal dissociation curves to lower urea concentrations. Incubation of CcO with increasing concentrations of GdmCl produces an analogous effect; however, the GdmCl-induced dissociation of subunits occurs at lower concentrations and with a narrower concentration range. Thermodynamic parameters for each subunit dissociation were evaluated from the sigmoidal dissociation data by assuming a single transition from bound to dissociated subunit. The free energy change accompanying urea-induced dissociation of each subunit ranged from 18.0 to 29.7 kJ/mol, which corresponds to 0.32-0.59 kJ/mol per 100 A(2) of newly exposed solvent-accessible surface area. These values are 30-50-fold smaller than previously reported for the unfolding of soluble or membrane proteins.

Figure 1

Subunit composition of CcO before and after exposure to various concentrations of urea. In each sample, dodecylmaltoside solubilized CcO was exposed to 0 - 7 urea for 2 hr at room temperature, the dissociated subunits and urea were removed by HiTrap Q anion exchange chromatography, and the subunit composition determined by either SDS-PAGE or reversed phase HPLC. Top Panel: SDS-PAGE analysis of the mitochondrial-encoded subunit composition CcO before and after exposure to 0, 3.0, 5.0 and 7.0 M urea. Bottom Panel: Reversed-phase C₁₈-HPLC subunit analysis of the nuclear-encoded subunit composition determined by analysis of 100μg (0.5 nmol) of the purified CcO using reversed-phase C₁₈ HPLC (refer to Methods for details). Elution peaks are labeled by roman numerals to indicate the elution peak corresponding to each of the nuclear-encoded subunits. The four elution profiles from the top down correspond to results obtained after exposure to 0, 4.0, 5.5, and 7.0 M urea, and are labeled accordingly. Prolonged incubation at high urea concentrations did not affect either the elution position or the shape of the elution peaks suggesting that covalent modification by urea break down products, e.g., cyanate, did not occur.

Figure 2

Concentration dependence of urea-induced dissociation of subunits from CcO. CcO was exposed to urea (0 - 7 M), and the subunit content determined after removal of urea and dissociated subunits by HiTrap Q anion exchange column chromatography (refer to Methods for details). Data were collected after exposure of CcO to urea for either 10 min (top panel), or 2 h (bottom panel) at room temperature. Five subunits dissociated with a sigmoidal dependence upon the urea concentration: III (grey-filled circles); Vb (black-filled circles); VIa black-filled triangles); VIb (unfilled triangles); and VIIa (unfilled circles). A full complement of the other eight subunits remained associated with the core of CcO, even at the highest urea concentration. Dissociation of each subunit could be fitted by non-linear regression analysis according to equation (5). For clarity, only the best-fit line through dissociation data collected for subunit VIIa are shown. Molar ellipticity data, θ_222nm (unfilled diamonds), is also included in each panel, which indicates that only minimal perturbation of the secondary structure occurs after exposure of CcO to 7 M urea for 10 min, or 4 M urea for 2 h at room temperature.

Figure 3

Urea-induced changes in the visible spectrum of CcO. Top Panel, absorbance spectrum of CcO after exposure to 6 M urea for 0 min (solid line), 10 min (dashed line), or 120 min (dotted-dashed line). Inset, corresponding difference spectrum relative to CcO exposed to urea for 0 min. Bottom Panel, second derivative spectrum of CcO after exposure to 6 M urea at room temperature for 0 min (solid line), 10 min (dashed line), or 120 min (dotted-dashed line). In each case spectra were acquired at 25 ° after CcO was incubated in 6M for the given length of time, and subsequently diluted 5-fold with 20 mM MOPS buffer, pH 7.2, to quench the dissociation reaction.

Figure 4

Concentration dependence of GdmCl-induced dissociation of subunits from CcO. CcO was exposed to GdmCl (0 - 2.2 M), and the subunit content determined after removal of GdmCl and dissociated subunits by HiTrap Q anion exchange column chromatography (refer to Methods for details). Data were collected and analyzed as described in Figure 2 for urea-induced dissociation of subunits from CcO. Data are shown for dissociation of subunits III (grey-filled circles), VIa (solid-filled triangles), VIb (unfilled triangles), and VIIa (unfilled circles), together with accompanying changes in the molar ellipticity, θ_222nm (unfilled diamonds).

Figure 5

Location of subunits within the three-dimensional structure of CcO that are either susceptible, or resistant to urea, or GdmCl-induced dissociation. The five subunits that dissociate (colored in this figure) are located near the dimer interface, and are in close contact with each other. This group of subunits is also the group that is sensitive to dissociation by elevated hydrostatic pressure [31]. Subunits that are resistant to dissociation, i.e., I, II, IV, Vb, VIc, VIIb, VIIc and VIII, are also clustered to form the catalytic core of CcO, (colored grey in this figure). Dimeric CcO can be considered to consist of three domains, two core domains that are urea and GdmCl resistant, and a third fragile, domain that bridges the other two (artificial separation of the CcO into these three domains is shown at the bottom of the figure). The third, fragile domain is roughly donut shaped with a hole in the center when viewed down the y-axis (yellow line). Dimeric CcO contains two copies of each subunit; therefore, the fragile domain is comprised of 10 subunits including two copies of subunits III (bright and lemon yellow), Vb (dark and light blue), VIa (bright and pale orange), VIb (bright and light pink), and VIIa (bright and dark cyan). All of the bright-colored subunits are associated with one CcO monomer, while all of the light, pale colored subunits are associated with the second monomer.