Metalloenzymes such as hydrogenases (H2ase), nitrogenase (N2ase), CO dehydrogenase (CODH) and methane monooxygenase (MMO) carry out some of the most fundamental energy converting reactions in nature at very high rates and with very high efficiency using earth abundant metals in their active sites. Despite the importance of these reactions in energy conversion, the mechanisms by which these enzymes carry out their reactions are poorly understood. Using a combination of biochemical, electrochemical and spectroscopic techniques our group is trying to learn how the active site structure and surrounding coordination spheres guide the mechanism of small molecule transformations in these enzymes. In particular, multiple spectroscopic techniques (UV-Vis, IR, Raman, EPR, MCD, Mößbauer and X-ray) are combined to provide a complete picture of the active site and interactions with the surrounding ligands.
A severe hurdle in studying many metalloproteins is often the low yield and lengthy procedure associated with purification from native host organisms. The enhanced yields, coupled with the simplicity of genetic manipulation, makes over-expression in recombinant hosts such as E. coli an attractive alternative. However, many metalloproteins cannot be easily produced in traditional E. coli systems and so the methods of expression or the hosts used must be further developed. Our group is developing stable recombinant expression systems for high yield production of metalloproteins, particularly sMMO, to facilitate sample intensive spectroscopic investigations such as X-ray absorption spectroscopy.
A key parameter for metalloproteins carrying out redox reactions is the redox potentials of their active site cofactors and the components of their electron transfer chains. How exactly the protein environment influences the cofactor redox potential, including the effects of direct metal ligands as well as more indirect influences such as hydrogen bonding, is a topic of intensive research. Our group is trying to understand these direct and indirect effects using simple iron-sulfur proteins (ferredoxins) as models. These proteins are easy to produce recombinantly in high yields and are easy to crystallize and study their structures. Furthermore, their redox potentials can be easily measured by protein film electrochemistry and their electronic structure probed by a range of spectroscopic methods.