Binuclear iron enzymes are widely distributed in nature and catalyze an extensive range of medically, industrially, and environmentally important reactions. These reactions all involve the activation of oxygen by very similar binuclear iron active sites. Intermediates can be trapped in these reactions and small binuclear iron designed peptides are available as models for these enzyme active sites. This research project involves using a wide variety of spectroscopic techniques combined with quantum mechanical calculations to understand the reaction mechanisms of this large class of enzymes and how apparently similar binuclear iron active sites perform very different functions. The achievement of these goals, in part, requires the development of new spectroscopic methods and definition of their information content from quantum theory. These studies define geometric and electronic structural contributions to O-O bond cleavage. Research also focuses on defining structural contributions to the complementary reaction of O-O bond formation by manganese cluster sites in photosynthesis.
Broader Impact The enzymes being studied are important in industrial synthesis, bioremediation, greenhouse gas elimination, iron metabolism, and as drug targets. The insights derived from these studies are being applied toward the development of new heterogeneous and bio-inspired catalysts. New spectroscopic methods, developed as part of this research program, have applications to a wide range of problems in chemistry, biology, materials and environmental sciences. The spectroscopic/electronic structure expertise of this research program is made available to the scientific community through a wide range of collaborations, which also leverage the impact of this research. This research provides students with a unique opportunity to use a variety of spectroscopic methods, develop new spectroscopic methodologies, and learn computational approaches to chemistry. The availability of such a wide range of techniques and theoretical insight allows students to learn to integrate these approaches to solve important problems in science. The students trained in the PI's lab (an equal gender mix) have become significant contributors in academics (> 40) and industry (> 50). This research group extensively participates in undergraduate teaching, mentoring women in science and outreach into the community to expose elementary school students in low-income areas and middle school females to science and careers in science. Through general seminars on spectroscopic methods, editing numerous books and thematic volumes, course readers, and significant participation on a range of editorial boards, this research program promotes the information content and utility of spectroscopic methods to problems in chemistry and biochemistry
This grant supports research into broad classes of enzymes containing nonheme diiron (NH 2Fe) active sites (figure, middle). Though NH 2Fe enzymes share similar structural characteristics, they display an extraordinarily diverse range of reactivities. Three examples depicted in the figure include methane monooxygenase (MMO), myo-inositol oxygenase (MIOX), and DNA protection during starvation proteins (DPS proteins). MMO catalyzes the oxidation of methane to methanol, a ubiquitous industrial reagent and fuel (figure, bottom left). MIOX converts myo-inositol to D-glucuronic acid in an important human metabolic pathway affecting mood, reproduction, and sensitivity to insulin (figure, bottom middle). DPS proteins prevent DNA damage from oxidative stress and are auspicious drug targets for anthrax and other pathogens (figure, bottom right). Our goals have been to: 1) determine the active sites of these enzymes and their relation to function; 2) elucidate the mechanisms by which these similar enzymes catalyze different reactions; and 3) further develop methodologies concomitant to these goals. To probe the environment of the enzyme active sites we have developed a variety of spectroscopic methodologies and coupled them to computations. The methods include magnetic circular dichroism (MCD) (figure, top left) and nuclear resonance vibrational spectroscopy (NRVS) (figure, top right), and then correlation to density functional theory (DFT) (figure, middle left). We also extend our studies to model complexes and de novo designed NH 2Fe proteins to systematically study structure-function relationships in diiron centers (figure, middle right). With these techniques we have made significant progress into advancing the goals listed above. Furthermore, we emphasize training scientists in this lab to become independent and productive members of the international scientific community post-graduation. We also strive to provide a foundational education to the local community through high school teacher/student mentorships and presentations at local schools. Focusing on our scientific goals, we have made great progress in investigating the oxygen-activating forms of these enzymes as well as several of their intermediates. Understanding the oxygenated intermediates of these enzymes is crucial in understanding their reaction mechanisms. Concerning goal 1), studies in our lab have led to a new reaction pathway with oxygen involving DPS proteins. In addition, we have made significant progress in our work with de novo NH 2Fe proteins synthesized to model MMO and similar enzymes. By systematically varying amino acids bound to the iron and characterizing the de novo mutant proteins, we have begun to develop fundamental insights connecting changes in structure to reactivity. Finally, we have further refined the structural understanding and mechanism of oxygen reactivity in MIOX and ferroxidase enzymes. Our studies involving goal 2) have focused on determining the oxygen-activation pathway for the protein ribonucleotide reductase (RR), yielding insights into DNA biosynthesis. A multitude of spectroscopic techniques have helped elucidate the structures of key intermediates during oxygenation. Quantum chemical calculations connected to our experimental data allowed evaluation of the energetics of oxygenation, refining the general understanding of the oxygenation pathway. Furthermore, our computations provide a predictive characterization of the electronic and geometric structures of key RR intermediates and thus suggest future directions for experimental studies. To further gain insight into NH 2Fe enzymes, we have developed new characterization methodologies involving NRVS and its coupling to quantum mechanical calculations. These techniques interrogate protein active sites unresolvable through other common methodologies. For example, intermediates found during oxygenation of RR and MMO show instability under laser irradiation, excluding resonance Raman characterization. These intermediates are, however, stable under NRVS experimental conditions and hence this technique offers a new avenue in determining the structure of active intermediates. To obtain a basis for the NRVS analysis of enzyme intermediates we have run NRVS on a number of structurally defined model iron/oxo complexes. Results from these experiments have expanded our understanding of NRVS data, and are being used in conjunction with NRVS data on NH 2Fe enzyme intermediates to elucidate their structures. Finally, NRVS combined with other spectroscopic methods have allowed us to probe NH manganese-iron proteins; our results further resolve the activation pathway for these enzymes. Our research investigates the molecular origins of enzymatic reactivity and affects many disciplines including drug design, bioremediation, metabolism, catalysis and biofuels. Our spectroscopic/electronic structure expertise has been made available through national/international collaborations which continue to expand to other universities. To facilitate scientific communication with the local community and promote scientific literacy, we have initiated a number of outreach programs involving our graduate students. One program includes a partnership with the Industry Initiatives for Science and Math Education (IISME), which invites high school teachers to work in our labs over summer and engage in scientific research. Our graduate students also present their research to high school students through the Inspiring Future Scientists program. This initiative is dedicated to communicating fundamental ideas in chemistry to young students as well as exposing them to university level research through shadowing.
