Defining the direct electron transfer connection between alternative complex III and cytochrome oxidase in Flavobacterium johnsoniae

Abstract

Alternative complex III (ACIII) is an enzyme of electron transport chains in some bacterial species. ACIII, like cytochrome bc enzymes, oxidizes quinol and transfers electrons from quinol to electron acceptors located outside the membrane. Various proteins can functionally link ACIII with other enzymes. The structure of ACIII from Flavobacterium johnsoniae suggests that in this bacterium the membrane-anchored mobile mono-heme cytochrome c domain (mdA) of the ActA subunit of ACIII provides means for its connection with cytochrome aa₃ oxidase. Here, using a recently-developed genetic system for ACIII, we revealed that ACIII mutant deprived of mdA does not exhibit electron transfer activity towards cytochrome aa₃ oxidase in the cells and in the isolated membranes. These results indicate that mdA is the only carrier of electrons between the pentaheme core of ActA and cytochrome aa₃ oxidase. In addition, we heterologously expressed and purified mdA and ActE (another mono-heme subunit of ACIII) from Escherichia coli to identify the redox midpoint potentials of the hemes in these two domains. The obtained values analyzed in the context of the whole titration profiles of native ACIII and ACIII deprived of mdA provide first insights into the arrangement of heme redox potentials in the seven-heme chain formed by the ActA/ActE assembly.