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Cytochrome c oxidase is the terminal enzyme in the respiratory chain of mitochondria and aerobic bacteria. This enzyme ultimately couples electron transfer from cytochrome c to an oxygen molecule with proton translocation across the inner mitochondrial and bacterial membrane. This reaction requires complicated chemical processes to occur at the catalytic site of the enzyme in coordination with proton translocation, the exact mechanism of which is not known at present. The mechanisms underlying oxygen activation, electron transfer and coupling of electron transfer to proton translocation are the main questions in the field of bioenergetics. The major goal of this work was to investigate the coupling of electron transfer and proton translocation in cytochrome c oxidase from Paracoccus denitrificans. Different theoretical approaches have been used to investigate the coupling of electron and proton transfer. This thesis presents an internal water prediction scheme in the enzyme and a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Two parallel molecular dynamics simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The average number of solvent sites in the proton conducting K- and D- pathways was determined. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping. The importance of the hydrogen bonding network and the possible coupling of local structural changes to larger scale changes in the cytochrome c oxidase during the catalytic cycle have been shown.
Nicotinic acid has been used in the clinical treatment of elevated blood lipid levels for over 50 years. Although it has a beneficial effect on myocardial infarction and blood lipid profiles, its widespread use has been hampered by side effects such as skin rashes and a burning sensation on the upper body. Since elevated blood lipid levels, especially ones of VLDL and LDL cholesterol are a frequent indication and high risk factor for coronary and cardiac diseases, finding a compound with an enhanced pharmacological profile, still holding the desired effects, but without inconvenient side effects, is a very appealing aim to many pharmaceutical companies. These efforts have already produced two marketed drugs, Acipimox and Acifran, but they have not been able to overcome the restrictions already imposed on the treatment by nicotinic acid. Although proposed long before, in the year 2000 the gene for the nicotinic acid receptor in mouse PUMA-G was cloned, and in 2003 the discovery of the genes HM74 and HM74A followed, which comprise the homologous low and high affinity receptors for nicotinic acid in humans. The discovery of this G Protein-coupled receptor target allowed a more directed approach for the search of alternative compounds. This work is the first report of the heterologous overexpression of the high affinity GPCR gene HM74A in the methylotrophic yeast Pichia pastoris. The protein product, NAR1, was pharmacologically characterized, and displayed a binding affinity of 224.8 nM to its ligand nicotinic acid, showing a similar activity profile compared to those displayed in human tissue, which were determined to be 60 nM to 90 nM. Additionally, inhibitory constants (Ki) for Acifran and Acipimox were determined to be 4.5 µM and 50.5 µM, respectively. Furthermore, the total yield of NAR1 reached 42 pmol/mg membrane protein, which corresponds to 0.4 mg of receptor produced per liter yeast culture, opening up the perspective of large scale protein production to facilitate high throughput screening drug discovery efforts and structural studies. In addition, NAR1 could be solubilized in n-decyl-β-D-maltopyranoside and purified to homogeneity after immobilized metal affinity chromatography and a second affinity chromatography step on immobilized monomeric avidin, yielding a single peak on gel filtration, while the purified receptor was able to bind ligand, as shown in NMR Saturation Transfer Difference (STD) measurements. It could be shown that NAR1 is desensitized by β-arrestin 1 in vivo in confocal microscopy studies on HEK and BHK cells. This finding provides a native binding partner for the stabilization of the receptor upon solubilization and purification. Finally human β-arrestin 1 could be produced as a constitutively active variant, comprising residues 1-382 in Pichia pastoris and Escherichia coli. The purified protein was used for in vitro binding experiments and shown to be capable of interacting with NAR1. Although the interaction and formation of the complex was only possible to a limited extent, it leaves open the perspective of crystallizing NAR1 in its active conformation, bound to nicotinic acid and β-arrestin 1.