Identification of Surf1 as an assembly factor of the cytochrome bc₁-aa₃ supercomplex of Actinobacteria

Respiration in aerobic Actinobacteria involves a cytochrome bc 1 -aa 3 supercomplex with a diheme cytochrome c 1 , first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of Cu A , Cu B , heme a, and heme a 3 . In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc 1 -aa 3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa 3 oxidase. In contrast to the wild type, a bc 1 -aa 3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa 3 oxidase.