Abstract
The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc1 and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation.
Highlights
The mitochondrion is the ‘‘energy factory’’ of eukaryotic cells as it provides ATP through the process of oxidative phosphorylation
We have previously reported that respiratory supercomplex factor 2 (Rcf2) purified from E. coli can form a dimer in dodecylphosphocholine (DPC) micelles where each Rcf2 monomer contains two flexible short helices (SHs) and five putative TM helices, including an unexpected charged helix at the C terminus, which is involved in dimer formation (Zhou et al, 2018a)
We present the solution structure of the Rcf2 dimer in DPC micelles, where the formation of the dimer is revealed to be mediated by hydrogen bonds and inter-monomer salt bridges occurring inside the micelle interior
Summary
The mitochondrion is the ‘‘energy factory’’ of eukaryotic cells as it provides ATP through the process of oxidative phosphorylation. In the mammalian mitochondrial inner membrane, four enzyme complexes, I (NADH-ubiquinone oxidoreductase), II (succinate dehydrogenase), III (ubiquinol-cytochrome c reductase, Cyt. bc1), and IV (cytochrome c oxidase, CytcO), constitute the electron transfer chain (ETC). In Saccharomyces cerevisiae, complex I is replaced by peripheral type II NADH dehydrogenases (see Refojo et al, 2019, for a recent review). The energy released in the redox reactions of the ETC is utilized to maintain a proton electrochemical gradient across the mitochondrial inner membrane, which is further used by complex V (ATP synthase) to generate ATP from ADP and inorganic phosphate. The supercomplex composed of a dimer of Cyt. bc (III2) and one (IV) or two (IV2) copies of CytcO was recently structurally solved to atomic resolution (Hartley et al, 2019; Rathore et al, 2019). The functional role of these supercomplexes is not clear (see Milenkovic et al, 2017), but they have been proposed to facilitate electron transfer to prevent generation of reactive oxygen species, and possibly to influence regulation of activities of these enzymes (Dudkina et al, 2010; Lobo-Jarne and Ugalde, 2018)
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