Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III2-IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII2 loops to conduct electron transfer between CIII2 and CIV.

Membrane proteins play important roles in various life processes, for example, those in the inner mitochondrial membrane (IMM), endoplasmic reticulum (ER) membrane, and plasma membrane (PM). Oxidative phosphorylation complexes, densely packed in the IMM are crucial for energy transduction in eukaryotes. We determined three entire II2III₂ IV2 supercomplex (SC) structures with 114 lipids at 2.1-2.4 Å resolution in Perkinsus marinus (P. marinus). The structures show a complete electron transfer pathway from complex II (CII) to complex IV (CIV). These architectures also reveal rotation states of the iron sulfur protein (ISP) in complex III (CIII), from one of which we observed two novel proteins that might impair the electron transfer. We also studied how the salt concentration and detergent affect the electron transfer. We determined the SC III₂ IV-cytochrome c (cyt. c) cryo-EM structure at 20 mM salt concentration condition. Together with kinetic study, these data implicate that multiple cyt. c are involved in electron transfer between CIII and CIV. Our kinetic studies of CIV also indicate a native ligand bound near its K proton pathway which could be removed by detergent, leading to an increase in electron transfer rate and the activity of the enzyme. Most biogenesis of integral membrane proteins in eukaryotes is done in ER, such as the amino acid permeases (AAP), which function as amino acid transporters in the PM. Its synthesis and functional folding in Saccharomyces cerevisiae (S. cerevisiae) requires an ER membrane-localized chaperone, Shr3. We utilized a yeast growth-based genetic assay, in conjunction with a split-ubiquitin yeast two-hybrid assay, to demonstrate the selective interaction between Shr3 and nested C-terminal AAP truncations. This interaction exhibited a distinct pattern, wherein it gradually intensified and then weakened as more transmembrane helices folded. The work presented in this thesis contributions to an increased understanding of the organization and function of SCs, the effects of protein subunits, salt concentrations, and detergents on electron transfer, as well as the mechanism of Shr3 on AAP folding in the ER membrane. Together, these works have shed light on the understanding of the structure and function of several membrane proteins.

Respiratory complexes have co-evolved into supercomplexes in different clades to sustain energy production at the basis of eukaryotic life. In this study, using cryogenic electron microscopy, we determined the 2.1 Å resolution structure of a 104-subunit II2-III₂-IV2 supercomplex from the parasite Perkinsus marinus, related to Apicomplexa, capable of complete electron transport from succinate to molecular oxygen. A feature of the parasite is the association of two copies of complex II via the apicomplexan subunit SDHG that interacts with both complexes III and IV and bridge the supercomplex. In the c1 state, we identified two protein factors, ISPR1 and ISPR2 bound on the surface of complex III, where Cytochrome c docks, acting as negative regulators. The acquisition of 15 specific subunits to complex IV results in its lateral offset, increasing the distance between the Cytochrome c electron donor and acceptor sites. The domain homologous to canonical mitochondria-encoded transmembrane subunit COX2 is made of three separate polypeptides encoded in the nucleus, and their correct assembly is a prerequisite for electron transport in the supercomplex. Subunits Cytochrome b and COX1 comprise a +2 frameshift introduced during protein synthesis by the mitoribosome. Among 114 modelled endogenous lipids, we detect a direct contribution to the formation of the divergent supercomplex and its functional sites, including assembly of CII and ubiquinone binding. Together, our findings expose the uniqueness of the principal components of bioenergetics in the mitochondria of parasites.

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane–localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone–substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3–AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3–AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.

Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we used cryo-EM and performed steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III-IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Previously unresolved negatively charged loops of CIII subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allowed us to identify water molecules in proton pathways of CIV and previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII loops to conduct electron transfer between CIII and CIV.

Cytochrome c oxidase (CytcO) is an integral membrane protein, which catalyzes four-electron reduction of oxygen linked to proton uptake and pumping. Amphipathic molecules bind in sites near the so-called K proton pathway of CytcO to reversibly modulate its activity. However, purification of CytcO for mechanistic studies typically involves the use of detergents, which may interfere with binding of these regulatory molecules. Here, we investigated the CytcO enzymatic activity as well as intramolecular electron transfer linked to proton transfer upon addition of different detergents to bovine heart mitoplasts. The CytcO activity increased upon addition of alkyl glucosides (DDM and DM) and the steroid analog GDN. The maximum stimulating effect was observed for DDM and DM, and the half-stimulating effect correlated with their CMC values. With GDN the stimulation effect was smaller and occurred at a concentration higher than CMC. A kinetic analysis suggests that the stimulation of activity is due to removal of a ligand bound near the K proton pathway, which indicates that in the native membrane this site is occupied to yield a lower than maximal possible CytcO activity. Possible functional consequences are discussed.