Separation and analysis of Bacillus subtilis respiratory chain complexes.

Bacillus subtilis is a Gram-positive bacterium with a respiratory chain embedded in the cytoplasmic membrane. The respiratory chain is bifurcated after menaquinol into a cytochrome b₆c + caa₃ branch and a branch with up to three quinol oxidases. The complexes that generate the proton gradient are b₆c, associated with caa₃ and aa₃ oxidase. The b₆c and caa₃ complexes form a supercomplex, and it is proposed to form respiratory strings in the membrane. There is still information missing about the quinol branch and if the primary oxidase quinol aa₃ is associated with the electron donor complexes. It is unclear whether succinate quinone reductase (SQR) can form associations with the quinol branch or the cytochrome branch. In this paper, we show the separation of an almost pure b₆c complex associated with cytochromes c₅₅₀ and c₅₅₁. We obtained a b₆c + caa₃ supercomplex of 600 kDa and SQR, aa₃, and NADH dehydrogenase by dodecyl maltoside solubilization and separation of the respiratory chain components by ionic exchange chromatography. We found that aa₃ does not associate with other complexes. SQR was associated with the b₆c complex in a mutant lacking aa₃. This association could facilitate electron transfer from SQR to menaquinone-7. The lack of associations between the abundant quinol oxidase aa₃ and other complexes is a feature we cannot explain yet.