Mechanisms of Assembly and Covalent Flavinylation in Complex II

Abstract

Complex II is an essential metabolic enzyme that functions in both the Krebs cycle and the electron transport chain in mitochondria, coupling succinate oxidation to fumarate with ubiquinone reduction to ubiquinol. Covalent FAD attachment to the flavoprotein subunit of this heterotetrameric complex is essential for succinate oxidation. Mutations that abrogate this covalent linkage result in pheochromocytoma-paraganglioma syndrome in humans although the underlying biochemistry is currently undefined. Several recently identified assembly factors are required for covalent flavin attachment, and mutations in the assembly factors can recapitulate clinical symptoms associated with Complex II deficiency. Understanding the mechanism of covalent flavin attachment is vital to understanding the function of Complex II and how this unique enzyme has adapted utilization of an FAD cofactor to couple two essential respiratory processes. Our work uses a combination of structural, biochemical and biophysical methods to investigate covalent flavinylation of Escherichia coli Complex II homologs, a process which may be mechanistically conserved from bacteria to mammals. Initial structural characterization of Escherichia coli Complex II homologs bearing mutations that prevent covalent linkage has provided insight into the potential role of inter-domain stability in the mechanism of covalent flavinylation. Additionally, site-specific photoaffinity crosslinking identified the binding location of the SdhE assembly factor on the E. coli Complex II flavoprotein, which suggests that this evolutionarily conserved assembly factor acts as a chaperone during covalent flavinylation.