Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure

Abstract

Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4 M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures.

Keywords: Chemical denaturation; Delipidation; Membrane protein stability; Protein-lipid interaction; Thermal denaturation.

Figure 1. MALDI-TOF/MS analysis of cytochrome c oxidase from Rhodobacter sphaeroides (upper panel) and single subunit I (lower panel)

One microliter aliquots were spotted on the MALDI target and overlayed with 1 µl of 10 mg/ml sinapinic acid in 50% acetonitrile/0.1% TFA. Spectra were acquired in linear mode and represent the average of 100 laser shots.

Figure 2. Structure of cytochrome c oxidase from Rhodobacter sphaeroides

Left – a side view showing all subunits, right – a top view showing helix arrangement and localization of molecules of phosphatidylethanolamines co-crystalized with the enzyme. Subunit I, red; subunit II, green; subunit III, yellow; subunit IV, grey. Four molecules of phosphatidylethanolamine localized between subunits III and IV in proximity of the subunit I are colored in cyan; two molecules of phosphatidylethanolamine localized in cleft of helixes of subunits III next to the subunit I are colored in dark blue. The structure is visualized with PyMol with Protein Data Bank entry 1M56 (Svennson-Ek et al. 2002).

Figure 3. Thin layer chromatography analysis

TLC analysis of standard phosphatidylcholine (PC) (left line) and phosphatidyethanolamine (PE) (right line) and extracted phospholipids from 2.5 nmoles delipidated CcO (middle line). TLC plate was sprayed with phosphor-molybdate solution to visualize spots containing phosphate (left plate) and then charred with the spray containing sulphuric acid to visualize carbon containing spots (right plate). On the plates, it is possible to detect also heme and used detergent dodecyl maltoside (DDM).

Figure 4. Spectral analysis of cytochrome c oxidase from Rhodobacter sphaeroides

Main panel: circular dichroism spectra of lipidated (thick lines) and delipidated (thin lines) CcO in the far-UV region indicate that delipidation does not affect secondary structure of the enzyme. Inset: nearly identical second derivative absorbance spectra of delipidated and delipidated CcO in the Soret region strongly suggest that delipidation does not affect heme region of CcO. The spectra of both lipidated and delipidated CcO were taken in 40 mM MOPS, pH 7.2, buffer containing 1 mg/mL dodecyl maltoside at room temperature. The protein concentrations were 0.8 µM and 5 µM at circular dichroism and absorbance spectroscopy measurements, respectively.

Figure 5. Temperature-induced denaturation of nondelipidated and delipidated Rhodobacter sphaeroides cytochrome c oxidase

Upper panel: Thermal denaturation of lipidated CcO followed by DSC (solid lines) and by ellipticity change at 222 nm (filled circles). Lower panel: Temperature-induced changes in ellipticity of lipidated (solid circles) and delipidated (empty circles) CcO followed by CD at 222 nm. Ellipticity changes were normalized to maximal differences. Inset: SDS-PAGE subunit separation. Line 1: lipidated CcO; Line 2 : lipidated CcO after heating to 80 °C; Line 3: delipidated CcO; Line 4: delipidated CcO after heating to 80 °C. After exposure of lipidated and delipidated CcO to high temperature the subunit III precipitated in both samples – line 2 and 4, respectively. The protein concentrations were 5 µM and 0.6–0.9 µM in DSC and CD, respectively. The enzyme was solubilized in 20 mM sodium phosphate buffer, pH 7.2, containing 2 mM of dodecyl maltoside.

Figure 6. Concentration-dependent effect of urea on the steady-state electron transfer activity of lipidated (solid circles) and delipidated (empty circles) Rhodobacter sphaeroides cytochrome c oxidase

Rbs-CcO was incubated at a concentration of 5 µM in 40 mM MOPS, pH 7.2, with 1 mg/ml DDM and varying concentration of urea. After 10 minutes incubation at room temperature, the effect of urea was stopped by dilution of the sample with 40 mM MOPS buffer following by incubation on ice. The enzyme activity was normalized towards the activity at 0M urea. Inset: Time dependence of normalized molecular activity of both lipidated (solid triangles) and delipidated Rbs-CcO (empty triangles) in 4M urea at room temperature. Delipidation of the protein did not affect the enzyme’s activity in the absence of urea. Activities of both lipidated and delipidated enzymes were 1450±150 s⁻¹ in the absence of urea.

Figure 7. Sedimentation velocity analysis

Lipidated (asterisks) and delipidated (empty circles) CcO in the absence of urea, and delipidated CcO after 10 minutes incubation in 4M (solid squares) and 6M urea (empty squares) at room temperature. Urea-induced heterogeneity of the protein indicates disintegration of CcO. Inset: Correlation between molecular activity and the amount of subunit II (empty triangles) and subunit III (solid triangles). The quantity of the subunits II and III were normalized to amount of the subunit I.