Enzymatic Activities ofIsolated Cytochrome bc1-like ComplexesContaining Fused Cytochrome b Subunits with AsymmetricallyInactivated Segments of ElectronTransfer Chains

摘要

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 intermonomerelectron transfer through the bridge effectively sustains the enzymaticturnover. The results fully support the concept that electrons freelydistribute between the four catalytic sites of a dimer and that anypath connecting the catalytic sites on the opposite sides of the membraneis enzymatically competent. The possibility to examine enzymatic propertiesof isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available forinvestigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives.