Living systems are the most sophisticated of chemical reactors. At any moment within a single cell several thousand processes, all critically inter-related, are being performed to ensure survival of the organism. This amazing feat is even more remarkable when you realise that most of these transformations take months, if not years, to occur when left to their own devices. A bag of sugar releases none of its energy when left in the kitchen cupboard!
Biology employs specialised molecular machines, called enzymes, to accelerate chemical reactions to the rates required to sustain life as we know it. Over 30% of these enzymes catalyse the movement of electrons from one molecule to another, one molecule being reduced while another is oxidised, and these are called redox enzymes. The molecules undergoing redox transformation are substrates and during the transformation they are temporarily attached to the enzymes at locations known as active sites. Often the substrate molecule binds to a metal atom built into the active site and a chain of metal atoms is employed to define a pathway through which electrons move between active sites. Thus redox enzymes are similar to the electrical appliances we encounter every day where metal wires shrouded by insulating polymer achieve directed electron movement and prevent short-circuiting.
By attaching redox enzymes directly to an electrode it is possible to quantitate the movement of electrons within them as an electrical current. But to understand their internal workings we also need a way to observe how substrate molecules and electrons pass through the enzyme, where they end up and how quickly they get there. We need a method that recognises when an electron or a substrate molecule are located on a metal atom. Fortunately, one of the metal centres most widely employed by redox enzymes has an intense colour that provides this information. This centre is the heme group, comprised of an iron atom surrounded by a special organic ring. The blood protein hemoglobin provides an example of how heme colour reports on heme status. Blood leaving the lungs is bright red in colour because an oxygen molecule is bound directly to the iron of hemoglobin whereas blood returning to the lungs has the purple colour of deoxygenated heme; the oxygen having been delivered to the bodies tissues and organs that are reliant on oxygen for their function.
In the laboratory, we record the colour of a heme in the form of an "absorption spectrum". This is a plot of how much light is absorbed by the heme at different wavelengths. This works well for one heme. However, some enzymes contain more than one heme and this makes it difficult to detect small differences in colour between the hemes. To overcome this we measure the spectrum using polarised light and a strong magnetic field in what is called an MCD experiment. This method amplifies the colour differences between hemes in the spectrum allowing us to see what is happening at individual hemes.
The work we proposed to do will allow us to perform MCD on enzymes attached to electrodes so we can determine which hemes are receiving electrons, in what order and how this changes when substrate binds to the active site and catalysis is initiated. By doing this, we will be able to examine in detail the internal workings of many enzymes. In this proposal we will focus on resolving the properties of two groups of enzymes. Those involved in cycling nitrogen and sulfur nutrients through the biogeosphere and those responsible for the ability of disease causing bacteria to respire in aerobic and anaerobic environments. In addition to resolving the properties of these enzymes our approach to wiring enzymes to electrodes may facilitate the exploitation of redox enzymes as molecular scale electronic devices, e.g. transistors and diodes, in the processors required for ultra-fast computing.