In-vitro, SDH5-dependent flavinylation of immobilized human respiratory complex II flavoprotein

Mitochondrial Complex II (Succinate: ubiquinone oxidoreductase) has a covalently bound FAD cofactor in its largest subunit (SDHA), which accepts electrons from oxidation of succinate during catalysis. The mechanism of flavin attachment, and factors involved, have not been fully elucidated. The recent report of an assembly factor SDH5 (SDHAF2, SDHE) required for flavinylation (Hao et al., 2009 Science 325, 1139-1142) raises the prospect of achieving flavinylation in a completely defined system, which would facilitate elucidation of the precise role played by SDH5 and other factors. At this time that goal has not been achieved, and the actual function of SDH5 is still unknown. We have developed a procedure for in-vitro flavinylation of recombinant human apo-SDHA, immobilized on Ni-IMAC resin by a His tag, in a chemically defined medium. In this system flavinylation has a pH optimum of 6.5 and is completely dependent on added SDH5. The results suggest that FAD interacts noncovalently with SDHA in the absence of SDH5. This system will be useful in understanding the process of flavinylation of SDHA and the role of SDH5 in this process.