Functional analysis and expression of the mono-heme containing cytochrome c subunit of Alternative Complex III in Chloroflexus aurantiacus.

The filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus possesses an unusual electron transfer complex called Alternative Complex III instead of the cytochrome bc or bf type complex found in nearly all other known groups of phototrophs. Earlier work has confirmed that Alternative Complex III behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. In this work, we focus on elucidating the contribution of individual subunits to the overall function of Alternative Complex III. The monoheme subunit ActE has been expressed and characterized in Escherichia coli. A partially dissociated Alternative Complex III missing subunit ActE and subunit ActG was obtained by treatment with the chaotropic agent KSCN, and was then reconstituted with the expressed ActE. Enzymatic activity of the partially dissociated Alternative Complex III was greatly reduced and was largely restored in the reconstituted complex. The redox potential of the heme in the recombinant ActE was +385mV vs. NHE, similar to the highest potential heme in the intact complex. The results strongly suggest that the monoheme subunit, ActE, is the terminal electron carrier for Alternative Complex III.