Search

Coenzyme Q (CoQn or ubiquinone) is an essential lipid molecule containing a redox-active benzoquinone head group and polyisoprenoid tail of varying length, denoted by n. The structural features of CoQ afford its ability to perform its role as a mobile electron carrier in the mitochondrial electron transport chain. Its synthesis is driven by the nuclear-encoded Coq polypeptides, several of which are required to assemble into a protein-lipid complex, the CoQ synthome. The formation of the CoQ synthome is coordinated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a membrane contact site that bridges the ER and mitochondria. CoQ deficiency results in various clinical manifestations, therefore it is imperative to have a holistic understanding of how CoQ is endogenously synthesized and distributed, and how these processes are regulated. We utilize Saccharomyces cerevisiae as a model organism given the high functional conservation of COQ genes and mitochondrial membrane contact sites with humans. Chapter 1 details an overview of the members required for CoQ biosynthesis and discusses the relationship between the CoQ synthome and ERMES. Chapter 2 focuses on the recharacterization of Coq10 using nonfunctional Coq10 isoforms. The COQ10 gene is positioned adjacent to MDM12, which encodes the cytosolic subunit of ERMES. Due to this arrangement, we discovered that deletion of COQ10 impacts MDM12 expression, resulting in diminished Mdm12 protein content and ERMES dysfunction. We use CRISPR-Cas9 to introduce mutations within COQ10 that preserve MDM12 expression, thereby maintaining ERMES. We show that Coq10 is not required for CoQ synthome assembly, and phenotypes associated with the coq10Δ mutant were due to the impact on ERMES. Considering ERMES may have a specific role in CoQ synthome assembly, Chapter 3 evaluates if ERMESΔ mutants can be rescued by deleting COQ11. The COQ11 gene product is proposed to be a negative modulator of CoQ synthome assembly, indicated by the ability of the COQ11 deletion to rescue complex stability in the coq10Δcoq11Δ double mutant. With Dr. Maya Schuldiner, we show that select ERMESΔ mutants can be rescued by deleting COQ11, indicating the role of ERMES in regulating CoQ synthome assembly can be circumvented when ERMES is absent. Chapter 4 examines how the expression of an artificial tether affects CoQ biosynthesis. Artificial tethers have been used to study membrane contact sites in vivo, and we report that the act of physical tethering does not bolster CoQ biosynthesis, and instead may impact trafficking and degradation. The results from Chapter 4 support the notion that ERMES has a direct role in CoQ synthesis and distribution, which an artificial tether cannot fulfill. Appendices I-III contain previous publications, including: characterization of the coq10Δcoq11Δ phenotype, an enzymology study from my undergraduate research published during my graduate program, and a structural analysis of clinically relevant single nucleotide variants in human COQ genes. Together, this work helps to clarify the roles of Coq10 and ERMES in the regulation of CoQ biosynthesis and distribution.

Abstract: A key enzyme in maintaining sulfide homeostasis is the membrane-associated flavoenzyme sulfide:quinone oxidoreductase (SQR) found in nearly all domains of life (except plants). SQR maintains a critical equilibrium between sulfide (H2S, HS- and S2-) and elemental sulfur (S0), coupling the oxidation of sulfide to the reduction of ubiquinone via a non-covalent FAD cofactor. SQR interacts with the membrane via two amphipathic helices and the Q-pool through a conserved hydrophobic domain. The active site of SQR includes three cysteines (Cys160, Cys356 and Cys128) and the FAD in close juxtaposition to the ubiquinone binding site. To understand the sulfide oxidation mechanism, we expressed the wild-type sqr gene from Acidithiobacillus ferrooxidans, as well as several variants of conserved, catalytically important amino acids in E. coli BL21(DE3) and purified soluble, active, His-tagged SQR. The purified wild-type SQR and the variants were subjected to extensive kinetic (pre-steady state and steady state) and structural analysis (X-ray crystallography). We also monitored SQR activity in vivo by detecting the H2S produced by growing E. coli transformed with wild-type and variant SQR. Our catalytic activity analysis and structural determination led us to propose two alternative mechanisms: (1) A nucleophilic attack mechanism that involves Cys356–S–S− as a nucleophile which attacks the C4A atom of FAD; or (2) A radical mechanism of direct electron transfer from Cys356 disulfide to FAD. The growing polysulfide is held between Cys160 and Cys356. The role of Cys128 (most likely in the form of a disulfide) is confined to the release of the polysulfur product. We further investigated the role of the FAD and the conserved Cys and His residues using a combination of kinetics and EPR spectroscopy. Using steady state kinetics of Na2S-dependent decylubiquinone (DUQ) reduction we measured a kcat of 6.5 s-1 and a Km (Na2S) of 3.0 M and a Km (DUQ) of 3.4 M. Variants of Cys160, Cys356 and His198 had greatly diminished DUQ reduction activity whereas variants of Cys128 and His132 were less affected. A neutral flavin semiquinone was observed in the EPR spectrum of SQR reduced with Na2S which was enhanced in the Cys160Ala variant suggesting the presence of a Cys356-Sγ-S-C4A-FAD adduct. Potentiometric titrations of the FAD semiquinone revealed an midpoint potential (Em) of -139 ± 4 mV at pH 7.0 in wild-type SQR. The Em of the FAD in SQRCys160Ala (Em= -135 ± 5 mV) is similar to that in wild-type SQR. We also combined computational docking and kinetic approaches to analyze quinone binding. SQR can reduce both benzoquinones and naphthoquinones. The alkyl side chain of ubiquinone derivatives enhances binding to SQR but limits the enzyme turnover. Pentachlorophenol and 2-n-heptyl-4-hydroxyquinoline-N-oxide are potent inhibitors of SQR with apparent inhibition constants (Ki) of 0.46 µM and 0.58 µM, respectively. The highly conserved amino acids surrounding the quinone binding site play an important role in quinone reduction. The phenyl sidechains of Phe357 and Phe391 sandwich the benzoquinone head group and are critical for quinone binding. Importantly, conserved amino acids that define the ubiquinone-binding site also play an important role in flavin reduction.

Coenzyme Q (ubiquinone or Q) is an essential redox-active, polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. Eleven genes products, Coq1-Coq9, Yah1 and Arh1, are required for Q biosynthesis in yeast Saccharomyces cerevisiae. Chapter 2 details the investigation of the biological function of Coq8 and its human homolog ADCK3. Expression of ADCK3 harboring an amino-terminal yeast mitochondrial leader sequence successfully rescued growth of coq8 mutants on non-fermentable carbon source, partially restored Q biosynthesis and the phosphorylation states of Coq3, Coq5, and Coq7. Chapter 3 investigates the use of over-expression of COQ8 as a tool to study Q biosynthesis pathway in S. cerevisiae. Over-expression of the Coq8 protein restores the steady state levels of the unstable Coq proteins. This stabilization results in the accumulation of several novel Q6 biosynthetic intermediates. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoci acid is used as a ring precursor in Q biosynthesis. Chapter 4 studies the use of para-aminobenzoic acid, and resveratrol as alternative aromatic ring precursors in Q biosynthesis in E. coli, S. cerevisiae, mouse and human cells. In contrast to S. cerevisiae, neither E. coli nor mammalian cells could utilize pABA as ring precursors in Q biosynthesis. However, E. coli cells labeled with 13C6-pABA generated several novel N-containing early intermediates, suggesting UbiA, UbiD,X, and Ubil are capable of using pABA as substrates. E. coli, S. cerevisiae, human and mouse cells cultured in the presence of 13C6-resveratrol were able to synthesize 13C6-Q. Thus, future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.

This dissertation presents a detailed computational investigation into the vibrational properties of quinones involved in solar energy conversion processes in photosynthetic reaction centers. In particular, we focus on the vibrational properties of the ubiquinone molecule that occupies the QA binding site in purple bacterial photosynthetic reaction centers. To provide a foundation upon which to base computational studies of pigments in protein binding sites density functional theory based calculations of the vibrational properties of neutral ubiquinone in the gas phase and in solvent were undertaken. From single point energy calculations it was shown that at least eight ubiquinone conformers, each with slightly different FTIR spectra, could be present in solvent at room temperature. The calculated and experimental spectra for neutral ubiquinone in solution are very different from the spectra associated with ubiquinone in the QA binding in purple bacterial reaction centers. For this reason an ONIOM method was undertaken in which the pigment was treated using density functional theory based methods while the protein was treated using molecular mechanics. The ONIOM calculations not only modeled the experimental QA FTIR difference spectra but also resolved the long standing issue of whether a very strong hydrogen bond exists between the bound ubiquinone and the imidazole nitrogen of a histidine residue (HisM219). To further validate the usefulness of the ONIOM approach experimental isotope edited FTIR spectra obtained using purple bacterial reaction centers with a range of chainless symmetrical quinones incorporated were modeled. Again, the agreement between the calculated and experimental spectra is outstanding. We also modeled the vibrational properties of the ubisemiquinone anion radical both in solvent and in the QA binding site. Vibrational modes of ubisemiquinone display a greater degree of mixing of the various molecular groups of the molecule. Nonetheless the calculated FTIR spectra for ubisemiquinone in solution and in the QA site agree very well with that found experimentally. Vibrational frequencies of ubisemiquinone obtained from ONIOM calculated Raman spectra also agree very well with that found in experimental resonance Raman spectra associated with the ubisemiquinone anion radical in the QA binding site.

Cancer is the second leading cause of death in the United States and its onset is highly incident in the lungs, with very low long-term survival rates. Chemotherapy plays a significant role for lung cancer treatment, and pulmonary delivery may be a potential route for anticancer drug delivery to treat lung tumors. Coenzyme Q₁₀ (CoQ₁₀) is a poorly-water soluble compound that is being investigated for the treatment of carcinomas. In this work, we hypothesize that formulations of CoQ10 may be developed for pulmonary delivery with a satisfactory pharmacokinetic profile that will have the potential to improve a pharmacodynamic response when treating lung malignancies. The formulation design was to use a vibrating-mesh nebulizer to aerosolize aqueous dispersions of CoQ₁₀ stabilized by phospholipids physiologically found in the lungs. In the first study, a method was developed to measure the surface tension of liquids, a physicochemical property that has been shown to influence the aerosol output characteristics from vibrating-mesh nebulizers. Subsequently, this method was used, together with analysis of particle size distribution, zeta potential, and rheology, to further evaluate the factors influencing the capability of this nebulizer system to continuously and steadily aerosolize formulations of CoQ₁₀ prepared with high pressure homogenization. The aerosolization profile (nebulization performance and in vitro drug deposition of nebulized droplets) of formulations prepared with soybean lecithin, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) were evaluated. The rheological behavior of these dispersions was found to be the factor that may be indicative of the aerosolization output profile. Finally, the pulmonary deposition and systemic distribution of CoQ₁₀ prepared as DMPC, DPPC, and DSPC dispersions were investigated in vivo in mice. It was found that high drug amounts were deposited and retained in the mouse lungs for at least 48 hours post nebulization. Systemic distribution was not observed and deposition in the nasal cavity occurred at a lower scale than in the lungs. This body of work provides evidence that CoQ₁₀ may be successfully formulated as dispersions to be aerosolized using vibrating-mesh nebulizers and achieve high drug deposition in the lungs during inhalation.

The interactions between mitochondrial function, reactive oxygen species (ROS), and lifespan are incompletely understood. Using the nematode C.elegans, this thesis attempts to clarify these interactions. The study focuses on the long-lived mutant clk-1 which is defective in synthesis of the electron carrier, ubiquinone. The work further investigates the role of ubiquinone on the following:1) Mitochondrial respiration.2) The site and rate of ROS production.3) ROS scavenging.4) The oxidation of mitochondrial proteins. Besides the studies of effects from different exogenous UQ, I also characterized the possible effects of the ubiquinone biosynthetic precursor, demethoxyubiquinone-9 (DMQ9) on mitochondrial electron transport and on phenotypes of clk-1. My thesis project integrates the comprehensive biochemical approach andC. elegans genetics to elucidate the mitochondrial effects on the variation in longevity. The results indicate that UQ10 extends the lifespan of clk-1 by loweringoxidative damage in mitochondrial proteins, but does not decrease ROS production. Moreover, DMQ9 inhibits electron transport from Complex I to III, which results in the defective Complex I-dependent respiration in clk-1. These results link the physiological changes within mitochondria caused by exogenous ubiquinone to aging and longevity.