Assembly and Activity of Respiratory Complex II in Nanolipoprotein Particles

Abstract

Mitochondrial respiratory Complex II (succinate:ubiquinone oxidoreductase) couples the citric acid cycle to the electron transport chain by oxidizing succinate in the matrix and passing electrons to ubiquinone in the inner membrane. Complex II is comprised of a soluble catalytic heterodimer (Sdh1/Sdh2) and a membrane-bound heterodimer (Sdh3/Sdh4). The structure of Complex II is well established; however, little is known about how the lipid environment regulates holocomplex assembly and activity. To address this question, we reconstituted Complex II from native biomembranes into nanoscale phospholipid bilayers (nanodiscs) containing a defined lipid content. We found that the dimeric phospholipid cardiolipin, the signature lipid of energy-conserving membranes, is critical for Complex II stability and function. First, the presence of cardiolipin in the bilayer promoted the interaction of the soluble and membrane-bound dimers. Second, cardiolipin was essential for enzymatic activity of the reconstituted complex and for curtailment of reactive oxygen species production. The function of cardiolipin could be partially compensated by the presence of phosphatidylglycerol, another phospholipid with an anionic headgroup; moreover, reducing the acyl chain lengths of cardiolipin used for reconstitution prevented its stimulatory effect on Complex II activity. Hence, both the headgroup and hydrocarbon chains of cardiolipin play important roles. Using this experimental platform, we have employed site-specific fluorescence labeling to address which structural elements of Complex II membrane subunits undergo conformational dynamics during assembly. Our results indicate that Sdh3 matrix-facing helix I, which interacts extensively with the catalytic dimer in the holocomplex, undergoes structural changes when reconstituted with Sdh4, but not Sdh4 homologs, suggesting that this helix may act as a conformational switch for downstream assembly steps (e.g., recruitment of the soluble dimer). Taken together, we show that Complex II is structurally dynamic during assembly and that its function is highly lipid-dependent.