Our group uses a wide range of spectroscopic methods coupled to calculations (density function and ligand field theories) to understand the electronic structure of metal sites and its contribution to physical properties and reactivity. Much of our research effort focuses on understanding key problems in bioinorganic chemistry.

The bioinorganic systems studied in our group primarily fall into four areas: electron transfer sites, copper active sites in biology, mononuclear non-heme iron enzymes, and binuclear non-heme iron enzymes. These systems are described below.

Electron Transfer Sites

Metal sites involved in biological electron transfer (ET) are very diverse and include blue copper, Cu_A, the 1-, 2-, 3-, and 4-iron sulfur centers in rubredoxin, ferrodoxins, and HiPIP, and the low-spin heme sites in cytochromes. Also relevant are heterometal-iron sulfur clusters and dithiolene-Mo oxo-transferases which are involved in multielectron small molecule activation, atom transfer, and proton coupled electron transfer (PCET). These active sites generally exhibit unusual spectroscopic features reflecting novel geometric and electronic structures that can make major contributions to function.

This research program has utilized a wide-range of spectroscopies combined with density functional calculations to understand these electronic structures, the origin of their geometric structures, and possible contributions to function. The spectroscopies utilized include multifrequency EPR, absorption, circular dichroism, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), resonance Raman (rR) excitation profiles, variable energy photoelectron spectroscopy (VEPES), ligand K- and metal K- and L-edge X-ray absorption spectroscopies (XAS). These spectroscopic methods have been used to evaluate electronic structure calculations (ligand field and density functional) and the combination is being applied to obtain new insight into copper and iron sulfur ET proteins, iron-heme sites, and the non-innocent nature of the dithiolene ligand found in molybdoenzymes.

Copper Active Sites in Biology

Copper proteins are involved in a wide range of biological oxidation-reduction processes. These include long-range electron transfer, dismutation of superoxide, reduction of nitrite and nitrous oxide, and reversible binding, transport, activation, and two- or four-electron reduction of dioxygen to peroxide or water that are coupled to substrate oxidation or proton pumping. This diversity can be attributed to the unique geometric and electronic structures of the copper active sites that are intricately tailored for their specific functions.

Biological copper active sites can be classified by their unique spectral features. These have been classically divided into type 1 “blue”, type 2 (T2) “normal”, and type 3 (T3) “coupled-binuclear” sites. However, recent studies on cytochrome c oxidase and nitrous oxide reductase have unveiled two novel classes of copper centers, the binuclear Cu_A and the tetranuclear Cu_Z sites, both of which have mixed-valent S_tot = 1/2 ground states. In addition, different types of copper centers are found in clusters, as in the trinuclear copper cluster site of the multicopper oxidases that are comprised of a T2 and a T3 site. Interestingly, the trinuclear copper cluster, upon reacting with O₂, produces a four-electron reduced oxygen intermediate that exhibits unusual spectral features that derive from the oxo-bridge mediated superexchange interactions among the three Cu(II) centers.

Our research covers a broad range of copper proteins, that mainly includes (a) coupled-binuclear Cu proteins involved in O₂ binding/transport and activation (hemocyanin, tyrosinase, and catechol oxidase), (b) non-coupled binuclear Cu proteins involved in O₂ activation and substrate hydroxylation (dopamine b-hydroxylase and peptidylglycine b-hydroxylating monooxygenase), (c) the multicopper oxidases involved in O₂ reduction (laccase, Fet3p, CueO, CotA, Ascorbate Oxidase, and Ceruloplasmin), and (d) the Cu_Z active site in the nitrous oxide reductase involved in the N₂O reduction. (T1 blue and Cu_A sites are mentioned in the ET section) Elucidation of the unique spectral and structural features of these active sites and their intermediates provides detailed insights into the nature of these sites that can be applied toward understanding their contributions to the unique catalytic functions in biology.

Mononuclear Non-Heme Iron

A large group of mononuclear non-heme iron enzymes bind and activate dioxygen to catalyze a variety of key biochemical transformations, including many of medical, pharmaceutical and environmental significance. These enzymes utilize high-spin Fe^II active sites and additional reducing equivalents from cofactors or substrates to react with O₂ to yield iron-oxygen intermediates competent to transform substrate to product. While Fe^II sites have been difficult to study due to the lack of dominant spectroscopic features, a spectroscopic methodology has been developed which allows the elucidation of the geometric and electronic structures of these active sites and provides molecular level insight into the mechanisms of catalysis.

These studies provide key insight into the mechanisms of oxygen activation, active site features that contribute to differences in reactivity and, combined with theoretical calculations and model studies, the nature of oxygen intermediates active in catalysis.

Binuclear Non-Heme Iron

Binuclear non-heme iron proteins have been shown to carry out a variety of different reactions through activation of dioxygen. These include O₂ binding (hemerythrin), activation for hydroxylation (methane monooxygenase, MMO), desaturation (Δ⁹desaturase, Δ⁹D), tyrosine radical formation (ribonucleotide reductase, RR by a proton coupled electron transfer (PCET) process), reduction by two-electrons (ferroxidase site of ferritin, and the related peroxidase site of rubrerythrin), and its four-electron reduction to water (rubredoxin: O₂ oxidoreductase, NO reductase, and the alternative oxidase in plants).

Despite a largely conserved ligand motif around the active sites in these enzymes, our studies utilizing absorption, resonance Raman, XAS, circular dichroism, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies applied in combination with density functional calculations provide significant insight into how geometric and electronic changes in the active site affect reactivity and allow for this diverse range of reactions to occur.

In addition to studying the resting states of these proteins, we are able to use rapid-freeze quench techniques to trap Fe₂^III-peroxide and high valent iron-oxo intermediates, as well as study other relevant oxygen-non-heme diiron model complexes and de novo designed protein analogues that have been synthesized. The spectroscopic data collected on these samples give great insight into the electronic structure and ligand environment of each Fe as well as the bridging between the irons. When combined with DFT calculations, we are able to obtain greater understanding of the structure/function correlations over the range of these binuclear non-heme iron enzymes and further elucidate their reactivity and reaction pathways.