The state of association of the cytochrome bc_1 complex from Paracoccus denitrificans in solutions of dodecyl maltoside

摘要

The cytochrome bc_1 complex is an intrinsic membrane protein of many respiratory chains. We studied the association state of solubilized cytochrome bc_1 from Paracoccus denitrificans by sedimentation equilibrium experiments in the analytical ultracentrifuge. Since the stability of the solubilized complex was only maintained in solutions containing low concentrations of the nonionic detergent n-dodecyl-β-d-maltoside (DDM), this detergent was also applied in the ultracentrifuge experiments. DDM is an unfavorable detergent in ultracentrifuge studies owing to its high density (ρ=1.23 g/ml), which strongly complicates "density-matching", the standard method for eliminating the contribution of protein-bound detergent to protein molar mass. Use of high sucrose concentrations for matching the DDM density is basically possible but probably induces significant changes in the protein's partial specific volume, v-bar; therefore we tried to decrease the necessary sucrose concentration by using mixtures of sucrose and 95% D_2O/5% H_2O (v/v). The effect of the solvent on &vmacr; was controlled by studying a related protein of known &vmacr;, cytochrome c oxidase, under identical conditions. In sedimentation equilibrium experiments the cytochrome bc1 complex behaved as an ideal homogeneous compound in the presence of 0.02% (w/v) DDM. Its molar mass was determined to be (240,000 ± 30,000) g/mol (mean and maximum error, respectively). Since the calculated mass of the protein protomer is 117,000 g/mol, the solubilized complex represents a dimer. Measurements in water-containing buffers, in the absence of sucrose, showed that the amount of DDM bound by the complex was (0.86 ± 0.12) g/g protein. A dimeric structure was already established for the much larger mitochondrial cytochrome bc1 complexes that had been crystallized. In the case of two other bacterial complexes experimental evidence points in the direction of dimers as well.