AscF in the mycobacterial CIII-CIV supercomplex lacks metal and nucleotide binding but links malate oxidation to...

In most Actinobacteria, the respiratory complexes CIII and CIV form an obligate supercomplex, but the exact subunit composition varies. Here, we have characterized AscF (MSMEG_4692), a subunit of the Mycobacterium smegmatis CIII-CIV supercomplex. We showed that AscF and the small, membrane-anchored AscG constitute a heteromeric TPM domain featuring a noncanonical topology. Biophysical analysis demonstrated that the isolated AscF/AscG module lacked intrinsic affinity for metals or respiratory nucleotides in vitro. Functionally, an ascF frameshift mutant exhibited abolished malate-dependent oxygen consumption and severe growth defects on nonfermentable energy sources. We conclude that AscF likely is not a sensor for metal ions or nucleotides but acts as an adapter subunit facilitating electron transfer from the tricarboxylic acid cycle to the mycobacterial respiratory supercomplex.