Correlating Current and Colour in Metalloenzymes: Cell development for time and concentration resolved Magnetic-CD Spectroscopy.

Approximately one in three proteins contains a metal, probably the best known example being hemoglobin which binds the transition metal iron as a heme that is responsible for the red colour of blood. Generally a protein will contain one or more transition metals when its task is to pass electrons between other proteins or when its substrate is a small inorganic molecule, an example being the dioxygen molecule that we inhale in order to live. In any case where a transition metal is involved we need specialised techniques to investigate how they work. Because the transition metals make the protein coloured one method to probe the nature of a metal site is to measure the absorption of light using electronic absorption spectroscopy. But the metals can also make the protein magnetic. If they are coloured AND magnetic then this allows us to use a technique called Magnetic Circular Dichroism spectroscopy, discovered by the British chemist Michael Faraday in 1845. This is similar to absorption spectroscopy but uses special polarised light and a strong magnetic field produced by a superconducting magnet. Magnetic Circular Dichroism spectra contain more features than absorption spectra and these identify ligands to the metal in addition to the number of electrons that are bound to it. Here we propose to develop novel equipment that will allow electron transfer within transition metalloproteins to be triggered from electrodes placed inside a superconducting magnet. In this way the resolving power of Magnetic Circular Dichroism spectroscopy will be harnessed to inform on the chemical nature of electron transfer and catalytic events inside transition metalloproteins.

Many proteins rely, for function, on the chemical reactivity of one or more bound transitions metal ions. This reactivity is intimately linked to the redox capabilities of such ions and, consequently, an elucidation of mechanism will always require a detailed understanding of these properties. This understanding is invariably gained through a combination of spectroscopic and electrochemical methods. In 'redox titrations' the electrochemical potential of the sample is varied systematically, using chemical reagents or electrodes/potentiostats, and the response of the cofactors is followed spectroscopically. Previously, we have developed 'MOTTLE' apparatus which allows such titrations to be followed using Magnetic Circular Dichroism (MCD), a powerful optical spectroscopic technique which can elucidate parameters such as the identity, spin-state, oxidation state and ligands of transition metal ions. MOTTLE requires that the electrochemical cell is small enough to mount in the narrow bore of the superconducting solenoid needed for the spectroscopy. Equilibration of solution state samples with the set potential of the electrode is slow and severely limits firstly the number of titration points obtainable in a set time and secondly the pathlength of the cell which in turn limits the strength of the MCD signals. We have recently demonstrated that these measurements can be performed using sample immobilised by adsorption onto tin oxide electrodes. We have also recently commissioned a new BBSRC-funded superconducting solenoid with a larger bore capable of accomodating larger cells. This proposal aims to significantly improve the capabilities of these methods by constructing a new generation of spectroelectrochemical cells which will (i) enable, for the first time, combined MCD and PFV (protein film voltammetry) and (ii) extend the range of transition metal ions which can be studied.