Enzymatic activities of isolated cytochrome bc₁-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains

Abstract

Homodimeric structure of cytochrome bc₁, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc₁-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc₁ with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc₁ with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc₁ from the mechanistic and physiological perspectives.

Figure 1

Asymmetric mutation patterns in cytochrome bc₁-like complexes containing fused cytochrome b subunit (B–B). The two halves of the fusion protein, each corresponding to one cytochrome b, are shown as white and gray rounded rectangles, respectively. Crosses indicate position of knockout mutations N and W, which refer to H212N and G158W point mutations in cytochrome b, respectively. Black arrows indicate functional branches. Black double arrow indicates electron entry point at the Q₀ site.

Figure 2

SDS-page analysis of B–B complexes containing mutations W and N in various asymmetric combinations. Samples of cytochrome bc₁ (WT) and various B–B complexes were isolated by Strep-tag affinity chromatography. M, marker (from IBA Biotechnology).

Figure 3

Spectroscopic proof of structural asymmetry imposed by mutations N and W in isolated B–B complexes. A–C compare spectra of pure B–B complexes isolated using Strep-tag affinity chromatography. A and B, optical spectra of hemes in isolated complexes reduced by dithionite (A) or ascorbate (B). Bottom spectra show the reference for cytochrome bc₁ containing H212N in cytochrome b (isolated by Strep-tag). C, X-band continuous wave EPR spectra of the FeS cluster in isolated complexes. All samples were treated with stigmatellin. B–B+G158W represents the sum of the normalized to g_y amplitude spectra of B–B and G158W. G158W is the reference spectrum for cytochrome bc₁ with G158W in cytochrome b (isolated by Strep-tag). Vertical dashed line shows the g = 1.909, which corresponds to the first inflection point of microwave absorption of g_y transition of the cluster for G158W mutant, while solid line shows g = 1.877, which corresponds to the second inflection point of absorption of g_y transition of the cluster of B–B form (as well the native cytochrome bc₁). Forms _WB–B, _W^NB–B, and _WB–B^N have the first and second inflections at g = 1.909 and g = 1.877, which confirms their asymmetry with respect to the mutation W. Form ^NB–B has the inflection points at the same g values as B–B. The dotted line approximates position of g_x transition (g = 1.775).

Figure 4

Comparison of enzymatic activities of cytochrome bc₁ and various B–B complexes. Plots show dependence of the turnover rate vs concentration of cytochrome c (Cyt c) in 50 mM Tris, pH 8, 0.01% DDM, 20 μM DBH₂. Fitting of the measured data points to the Michaelis–Menten kinetics yielded the values of V_max listed in Table 1. Broken line shows the estimated level of activity of _WB–B^N in which heme b_L–b_L electron transfer is assumed not to occur on a catalytic time scale. It was calculated as 0.5 × (V^W_max + V^N_max), where V^W_max and V^N_max denote the values of V_max determined for the isolated cytochromes bc₁ containing mutations G158W and H212N, respectively.