Principal Investigator Stephen Lippard
Polymetallic centers occur in numerous metalloproteins where they carry out a variety of functions including hydroxylation of methane, the generation of amino acid radicals, hydrolysis of phosphate esters, and dioxygen transport. How do two or more metal atoms in close proximity in these proteins work in concert to carry out their biological function? How do we know the coordination environment of such polymetallic species in biological molecules? To help answer these questions, we synthesize and characterize well-defined model compounds and compare their properties with those of their biological counterparts. Physical and biological studies of the proteins are also undertaken. Recently our attention has focused on systems in which one or more carboxylate groups link two metal ions. Proteins in this category include soluble methane monooxygenase (sMMO), an amazing biomolecular machine that converts methane and dioxygen selectively to methanol and water, and the related protein toluene/o-xylene monooxygenase (ToMO). Related eukaryotic enzymes from mammalian and other sources perform chemistry that is important for mitochondrial metabolism. We have now proved these systems to have analogous carboxylate-bridged diiron centers. The chemistry employed by this superfamily of enzymes involves activation of dioxygen at reduced, diiron(II) centers followed by dioxygen activation and substrate oxidation. Transient diiron(III) peroxo or diiron(IV) oxo species are responsible for the hydrocarbon oxidation step. We use chemical, EPR, Raman, NRVS, and optical spectroscopic, redox, EXAFS, X-ray crystallographic, NMR, Mössbauer and magnetic, and freeze-quench and stopped flow kinetics techniques to determine the how the metal ions activate O2 in the proteins. Intermediates are probed by freeze-quench spectroscopic and low temperature X-ray crystallographic methods. Further information about reaction intermediates and transition states are provided by density functional QM/MM theoretical studies in collaboration with the Friesner laboratory at Columbia University.
Studies with synthetic models focus on the use of sterically hindered carboxylate, macrocyclic, and preorganized ligands to afford complexes that best mimic the stoichiometric and functional properties of the enzyme active sites. With the use of the models we now have carboxylate-bridged diiron systems that can perform O2 activation, C–H bond hydroxylaton, and catalytic oxo transfer reactions. Important design features are the positioning of two N-donor ligands syn to the Fe–Fe vector and sufficient flexibility to allow for carboxylate shifts during the reaction chemistry.
Apart from this chemistry at the hydroxylase components of the enzymes we are investigating the structures and functions of the reductase, coupling, and other proteins in the systems alone and in complexes with the other components. Solution NMR spectroscopy has been used to determine the structures of the coupling protein, and the ferredoxin and flavin components of the sMMO reductase. The electron transfer reactions are also being studied. Access of four substrates, namely, protons, electrons, dioxygen, and a hydrocarbon, to the active sites of these enzymes are being extensively investigated. Site-directed mutagenesis is employed to interrogate functions of amino acid side chains in the second and third coordination shells of the hydroxylase active sites and to investigate protein-protein interactions important for function.