This award in the Chemistry of Life Processes (CLP) program in the Division of Chemistry at NSF supports a collaborative project between Professor Alan A. DiSpirito at Iowa State University and Professor Eckard Munck at Carnegie Mellon University to carry out fundamental studies aimed at elucidating the structure of the active site and the reactivity of the so-called particulate form of methane monooxygenase (pMMO). This is a ubiquitous bacterial enzyme that mediates the conversion of methane to methanol and thus plays a key role in the global carbon cycle. The selective oxidation of methane to methanol using mild reaction conditions is one of the most important industrial processes pursued by organometallic chemists and could be a direct application of the structural elucidation of pMMO's active site. The project includes the extensive use of Mössbauer spectroscopy to characterize the purported diiron center in pMMO, the identification of catalytic and electron transfer centers using Electron Paramagnetic Resonance (EPR) spectroscopy, and a mechanistic investigation of methane oxidation. This is an eminently interdisciplinary project at the interface of biochemistry, microbial physiology, and biophysical chemistry and will expose undergraduate and graduate students to a variety of techniques, ranging from protein purification to EPR spectroscopy.
The broader impacts of the proposal are focused on the training of students, including members from underrepresented groups and undergraduate students from the University of Wisconsin-Eau Claire, a Predominantly Undergraduate Institution.
Dioxygenases and hydroxylases are enzymes that activate oxygen to perform a large variety of molecular transformations in biosynthetic and biodegradative biochemical reactions. Many of these enzymes contain one or two iron atoms that are at the heart of these transformations. Mössbauer spectroscopy is a technique superbly suited to probe in detail the reactions taking place when substrates (the compounds to be transformed) and molecular oxygen bind to the iron atoms. For some of these enzymes, we and our collaborators have mapped in some detail short-lived (about one hundreds of a second) reaction intermediates and provided spectroscopic parameters which give insight into the way these enzymes work. Our research is carried out at the interface of physics, chemistry, biochemistry and genetics, giving our students and postdoctoral researchers a broad perspective in interdisciplinary work. The work reported below was performed with two collaborating groups at the University of Minnesota who were in charge of the chemical and biochemical aspects of the work. Our work has been published in leading journals and reported at various meetings. For instance, at the Penn State 2nd Bioinorganic Workshop the Principal Investigator and his student each have given 75 min tutorials to 60-80 graduate students from the US and Europe, describing the methodology of our approach. On the present grant we have worked on six different projects which share the theme of oxygen activation (transforming molecular oxygen into a reactive species). One of the projects involves the study of 2,3 homoprotocatechate dioxygenase (HPCD). For this enzyme we have characterized various catalytic intermediates, and in order the trap other suspected intermediates states we studied the reaction with "slow" substrates and/or used variants of the enzyme for which critical amino acids have been mutated, allowing us assess their importance. The particular mutation used, labeled Y257F, slows down some steps about a 100-fold which allowed us to asses two states not seen so far. We have characterized the variant enzyme and its complex with substrate in great detail, obtaining parameters that can serve as valuable benchmarks for quantum chemical calculations. Methane monooxygenase (MMO) is an oxygen activation enzyme containing a dinuclear iron center at the catalytic site. The enzyme converts methane to methanol, a very difficult conversion of utmost importance. In the past we have characterized some short-lived intermediate states for this enzyme, among them intermediates P and Q. In P the oxygen has already been activated by the enzyme to the peroxide level, and Q contains two tetravalent irons, di-iron(IV), and a highly activated oxygen. We have shown here that there is another intermediate state, P*, preceding P, and that this intermediate is in the diferrous oxidation state. The electronic structure of Q has been of intense interest ever since this state was reported by us in 1993. In this grant we have approached this problem from a different angle, namely by studying a synthetic di-iron complex which could be manipulated into the di-iron(IV) state. This complex is based on the coordinating ligand tris(2-pyridylmethyl)amine (TPA). We were able to study this system in various high oxidation states of the iron, and observe a million-fold increase in oxygen transfer and hydrogen abstraction reactivity. Most importantly, as reported for this grant, we were able to generate and characterize two additional novel di-iron(IV) states, bringing the number of di-iron(IV) states for this particular iron-TPA complex to four (these differ by geometrical configurations and magnetic couplings between the irons). One of the di-iron(IV) states is diamagnetic and has Mössbauer parameters like intermediate Q. We learned in this project, and this has general importance, that diiron(IV) complexes with low-lying electronic states can assume various configurations and spin states which strongly impact chemical reactivity and magnetic properties. The two irons in the TPA complex are linked by an oxo group that mediates exchange coupling between the two irons. Study of this TPA complex revealed that this exchange coupling can cause a spin transition from low-spin to high-spin at one of the iron sites, a phenomenon, to our knowledge, not reported before.
