|
Most chemical and biological reactions occur in solutions, where reactants can move freely (diffuse) until they collide and reaction takes place. In some cases, however, products of initial reaction are so unstable that readily engage in secondary reactions and convert into other forms. Extreme reactivity of radical species is an illustrative example, where radicals will quickly react with any available substrate or with each other and thus do not accumulate to any significant extent.
A key role of enzymes in metalloradical catalysis is to control reactivity of radicals by shielding them from all but specific substrates. Protein-bound radical species are practically immobilized in controlled protein environment, although water-soluble enzymes themselves can diffuse in cytosol. Other, membrane-bound enzymes are anchored or imbedded in biological membranes. Imbedded molecules can move along membrane surface, but much slower than soluble molecules.
We study spectral properties of isolated radical species as models for radicals involved in biological catalysis. High reactivity of free base radicals leads to very short lifetimes at ambient conditions (few milliseconds), which significantly hinders their thorough characterization. The lifetime of radical species can be extended if their mobility is restricted. We extensively use classical method of immobilization in frozen glasses at cryogenic temperatures. Unfortunately glassing methods have several drawbacks, including limited range of cryoprotectants, inaccessibility if sample, and conditions that are typically differ significantly from physiological.
Following the strategy of Nature, we are developing alternative methods of immobilization of small molecules. One of approaches involves binding of radical precursors to inert metal ions, such as Zr4+. In the presence of phosphate ions, zirconium readily form an extensive layered structures that are completely insoluble. Zirconium ions imbedded in lattice can still bind negatively charged ligands, particularly carboxylic acids, which are present in every amino acid and are very common in proteins. We are currently optimizing methods of irreversible binding of carboxylates to zirconium under relatively mild conditions. Once immobilized, carboxylates and amino acids would form more stable radicals at ambient conditions and subjected analysis by our spectroscopic methods. We are also exploring the possibility of secondary immobilization of zirconium phosphate lattice on the surface of electrodes for reversible electrochemical oxidation of model molecules.
Another approach for immobilization of molecules is directly inspired by the key cell structure - biological membranes. Lipids in water can form small cell-like vesicles, whose walls consists of a single lipid bilayer surrounded by water. Depending on the properties of lipid and temperature, bilayer can stay liquid or solidify, controlling diffusion of any anchored or imbedded molecule. Like with zirconium lattice, lipid bilayers can exist in a suspension or attached to a surface. On clean solid surfaces lipids can spontaneously form continuous bilayers, called supported bilayers. If solid surface is an optically transparent electrode, redox states of molecules imbedded in lipids can be directly manipulated electrochemically. Lipid bilayers are less robust than zirconium lattice, but they provide a flexible framework for immobilization of a broad range of biomolecules. Furthermore, lipid environment closely resembles that of biological membranes for such transmembrane enzymes as cytochrome c oxidase, for example.
|