new approaches for fresh perspectives on quinol/quinone oxidoreductases

All living cells are surrounded by a thin membrane that shields and separates the inside of these cells from their surroundings. In more advanced organisms, organelles are located inside the cell with specific functions. Also these organelles are separated (compartmentalised) from the rest of the cell by membranes. These thin membranes contain many proteins that actively transport compounds, like nutrients and salt, specifically across the membrane. Consequently, the concentration of many compounds is different on the inside of the membrane compared to the outside. These gradients play a crucial role in biology and many reactions in the cell are dependent on them, like photosynthesis and metabolism. Some of these proteins actively 'pump' protons across the membrane using energy that is released from electrons that are formed when sugars and fats are 'burned' by the cell. These electrons do not flow freely in the cell, but are attached to small molecules which 'float' in the membranes of the cell. These molecules are called quinones or co-enzyme Q. This proposal aims to develop a new tool with which we can study the proteins that are located in the membrane and react with quinones. Why do we want to learn more about these proteins? These proteins are involved in many important reactions. For instance, in bacteria they are responsible for all reactions involving nitrogen and carbon dioxide and therefore control how these elements are recycled in our atmosphere. In humans, similar proteins are involved in the burning of sugars and fat and the production of energy; Any problems with these proteins and we become ill. Finally, quinones themselves are 'anti-oxidants' and known to take away so-called 'radicals' which are thought to play an important role in diseases and aging. When we study the structure and function of proteins and quinones in the lab, they are normally taken out of the membrane and thus the environment of these proteins and quinones is changed a great deal. This is done because membranes do not dissolve in water and most of our experiments are performed in water; we thus need to take the membrane away. However, in this proposal we aim to develop a new tool that allows the study of membrane proteins and the quinone in their natural environment, the membrane. For this to be achieved, we will first place a 'membrane protein' that normally receives or gives electrons to the quinones on a solid surface. This solid is conducting (like metal wires) and we will carefully control the properties of the surface so that it will be possible to give or take electrons to or from the protein. We will then place a membrane on top of the proteins and this membrane will contain quinones. If everything works as we think it will, the protein will give or take electrons to or from the quinones. As the transfer of electrons is nothing more than electrical current, we can measure very accurately how fast these electrons are passed from the surface to the proteins and into the quinones (or the other way around). Once this system is complete, we can use these surfaces to 'interrogate' these membrane proteins in almost the same membrane environment they encounter in the cell. By studying these proteins we will thus learn more about how they function inside their natural membrane.

Quinone oxidoreductases (QORs) play central roles in respiration and photosynthesis with additional roles in anti-oxidant production and biosynthesis. However, when compared to our understanding of globular enzymes, relatively little is known about the kinetics of enzyme-catalysed QH2 ? Q transformations. In large part this is a consequence of constraints on diffusion and solubility that result from membrane environments that have proved difficult to mimic and manipulate reproducibly until recently. Here, we will combine two 'tools' - solid-supported membrane technology and protein-film voltammetry - to develop new methods for quantitative analysis of QOR catalysed transformation of lipophylic substrates. Studies with members of a widespread family of respiratory QH2 dehydrogenase will provide proof of principle for methods that will be ultimately be applicable to many QORs. Three strategies will be compared for their ability to support electrochemical characterisation of QOR activity in a membrane environment supported by a graphite or gold electrode, i) proteoliposome adsorption, ii) vesicle adsorption on adsorbed protein films and iii) vesicle fusion, i.e., planar membrane formation, on adsorbed protein films. Voltammetry will define the stability and electroactive coverage of the films, the reduction potentials and interfacial electron transfer rates for the adsorbed proteins and their QOR activities. Where films are formed on gold electrodes complementary information will be provided from other methods. Surface plasmon resonance, atomic force microscopy and quartz crystal microbalance methods will define the total amount of adsorbed protein for comparison to the amount of electroactive protein. Surface-enhanced resonance Raman spectroscopy and/or surface-enhanced Fourier transform infrared spectroscopy will inform on structural differences between assemblies.