The roles of SDHAF2 and dicarboxylate in covalent flavinylation of SDHA, the human complex II flavoprotein

Abstract

Mitochondrial complex II, also known as succinate dehydrogenase (SDH), is an integral-membrane heterotetramer (SDHABCD) that links two essential energy-producing processes, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. A significant amount of information is available on the structure and function of mature complex II from a range of organisms. However, there is a gap in our understanding of how the enzyme assembles into a functional complex, and disease-associated complex II insufficiency may result from incorrect function of the mature enzyme or from assembly defects. Here, we investigate the assembly of human complex II by combining a biochemical reconstructionist approach with structural studies. We report an X-ray structure of human SDHA and its dedicated assembly factor SDHAF2. Importantly, we also identify a small molecule dicarboxylate that acts as an essential cofactor in this process and works in synergy with SDHAF2 to properly orient the flavin and capping domains of SDHA. This reorganizes the active site, which is located at the interface of these domains, and adjusts the pKa of SDHAR451 so that covalent attachment of the flavin adenine dinucleotide (FAD) cofactor is supported. We analyze the impact of disease-associated SDHA mutations on assembly and identify four distinct conformational forms of the complex II flavoprotein that we assign to roles in assembly and catalysis.

Hepatic Suppression of Mitochondrial Complex II Assembly Drives Systemic Metabolic Benefits

“…In addition, mRNA levels of other mitochondrial complex subunits were also unaltered by ADF (Figure 1i ). Instead, the protein levels of SDHAF2 and SDHAF4, two assembly factors initiating the assembly of SDH complex by promoting SDHA maturation and binding with SDHB, [ 25 , 26 ] were significantly altered by ADF (Figure 1g,h ). Even though SDHAF2 protein was slightly increased, the binding activity of SDHAF2 with SDHA and SDHB was not affected (Figure 1j ).…”

“…Yet, functional complex II still requires sequential assembly with help of assembly factors. [ 41 ] SDHAF2 was suggested to mediate the maturation of SDHA, [ 25 ] which was further promoted to assemble with SDHB by SDHAF4 to initiate the assembly of complex II. [ 26 , 42 ] Thereby SDHAF2 and SDHAF4‐mediated activities are the key steps for functional complex II assembly.…”

Calpain-mediated protein targets in cardiac mitochondria following ischemia–reperfusion

“…Deletion of CPNS1 leads to decreased succinate dehydrogenase assembly factor 2 (SDHAF2) (Table 4 ) that is essential for the assembly of succinate dehydrogenase 61 . These decreased proteins may explain why deletion of CPNS1 does not improve OXPHOS with succinate as a complex II substrate in mitochondria during ischemia–reperfusion.…”

YgfY Contributes to Stress Tolerance in Shewanella oneidensis Neither as an Antitoxin Nor as a Flavinylation Factor of Succinate Dehydrogenase

“…Structure investigations show that Gly in the RGXXE motif of the flavinylation factor of SDH plays the exact same role in all investigated organisms crossing three kingdoms. Gly in RGXXE motif forms a critical hydrogen with SdhA in SDH in E. coli and humans [32,33]. Moreover, Gly in RGXXE of SdhE(CptB) can also form a hydrogen bond with FrdA in the fumarate reductase complex and is indispensable for FrdA flavinylation in E. coli [34].…”