Spectroscopic Definition of Electronic Structure and Contributions to Reactivity of Binuclear Non-Heme Iron Enzymes

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Binuclear non-heme ferrous active sites are involved in a wide-range of reactions with dioxygen including O2 binding (hemerythrin), activation for hydroxylation (methane monooxygenase, MMO), desaturation (desaturase), 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). Structures exist for many of these proteins in at least one oxidation state, Fe2III-peroxide and high valent iron-oxo intermediates have been trapped, and relevant oxygen-non-heme diiron model complexes and de novo designed protein analogues have been synthesized. Spectroscopic methodology emphasizing variable-temperature variable-field magnetic circular dichroism (VTVH MCD) has been developed to probe the biferrous active sites and analyze the excited and ground state interactions in terms of the geometric and electronic structure of each iron and the bridging ligands. VTVH MCD combined with other spectroscopies (resonance Raman excitation profiles, X-ray absorption, EPR, etc.) have further been applied to define the geometric and electronic structures of a number of the oxygen intermediates in the proteins and models and their relevance to reactivity. Combining these data with electronic structure calculations (DFT) has provided significant insight into geometric and electronic structure/function correlations. Research now focuses on using this spectroscopic/computational methodology to structurally define the intermediates in the reaction coordinates of RR and MMO. These studies will define the structural changes of the biferrous resting state associated with activating the site for O2 reactivity, determine geometric and electronic structural differences that contribute to differences in substrate reactivity (desaturation, hydroxylation and PCET), and generally evaluate geometric and electronic structure differences over the range of proteins and enzymes listed above (and de novo designed analogues) which contribute to their different roles in O2 biochemistry. Iron enzymes and proteins play key roles in many important biological functions. The focus of this research is to understand the structure and function of binuclear non-heme iron enzymes, providing molecular level insight into reaction processes and catalysis. The enzymes studied are important in catalytic processes, biotechnology, environmental regulation, and DNA biosynthesis. The scientific methodologies developed as part of this study have applications to a variety of important classes of metal sites in biology and catalysis. Postdoctoral associates, graduate students and undergraduates involved in this research are well trained in a wide range of spectroscopic methods, quantum chemistry calculations and enzymology, and have gone on to become significant contributors in academia and industry. The instrumental and theoretical resources available in these labs for the study of binuclear active sites have been made accessible to the bioinorganic community through a range of collaborations which also leverage these resources to enhance impact on the field. The PI has also attempted to enhance the general awareness of the importance and information content of the different spectroscopic methods in bioinorganic chemistry through organizing symposia, books, overview articles and presentations of general lectures and lecture series on this topic. There have also been a wide-range of sabbatical visitors spending time in the PI's labs at Stanford to further their knowledge in this area ranging from well established bioinorganic/biophysical chemists, to a local high school chemistry teacher and an under-represented minority Professor from an undergraduate college interested in enhancing research. This project is jointly funded by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Inorganic, Bioinorganic and Organometallic Program in the Chemistry Division.