Biological Transition Metals

Our program is devoted to understanding the function of biologically central transition metals. We here focus on three key roles of transition-ion centers, and have assembled outstanding multidisciplinary teams to attack them. The approaches to each incorporate a suite of advanced paramagnetic resonance techniques, EPR/ENDOR/ESEEM, many of which we have developed. (a) 'Radical-SAM (S-adenosyl methionine)' Enzymes: This enzyme superfamily is Nature’s most widespread means of performing essential radical-based chemistry. (i) With Broderick, we have demonstrated that throughout the superfamily, reductive SAM cleavage generates an organometallic intermediate, , central to catalysis. We will probe the properties and reactivity of  through EPR/ENDOR studies of multiple ’s, in parallel with studies of synthetic -analogs prepared by Suess. (ii) We revealed that regioselective cleavage of the SAM S−C5’ bond to generate  upon SAM reduction is Jahn-Teller (JT) enabled and active-site controlled. To understand this phenomenon we will study it with a selected suite of SAM analogues bound in a correspondingly selected suite of RS enzymes, while computationally exploring the fundamentals of the process with Mosquera. (iii) We will expand the study of the RS catalytic mechanism, examining substrate transformations by epimerases and spliceases. (b) Mechanism of Nitrogenase activation: With Dean, Seefeldt, and Raugei we have revealed how the nitrogenase MoFe protein is activated to carry out perhaps the most challenging chemical transformation in biology, the reduction of the N≡N triple bond, and have shown that the alternative V- and Fe-nitrogenases employ the same mechanism. This latter finding will enable us to explore the structure of nitrogenase intermediates throughout the entire catalytic cycle, using 1,2H/14N ENDOR of substrates. A remarkable achievement of Dean enables us to monitor the 13C ENDOR of carbide central to the FeMo-co active center, as well. This study is enhanced through the use of site-selectively 57Fe-labeled FeMo-co in enzyme prepared by Suess, a major advance in integrating structure and function. (c) in vivo Mn2+ Speciation: We earlier established that EPR/ENDOR/ESEEM provide an otherwise unavailable means of characterizing Mn2+ complexes in live cells. Our collaboration with Daly now has shown that Mn2+ speciation is the strongest biological indicator of cellular resistance to ionizing radiation (IR) throughout the tree of life. In the coming period we will explore a discovery of and correlation between in vivo Mn2+ speciation, IR resistance, and aging, and test the hypothesis that our spectroscopies can be used to devise optimized radiotherapy regimens for human tumors. In a dramatic new venture, a collaboration with O’Halloran surprisingly indicates that Mn2+ ions play an important role in fertilization of amphibian oocytes, and we will broaden and expand our studies of Mn2+ speciation by examining fertilization in mammals. Synergy: Each of the enzyme systems addressed a problem of fundamental importance, while the diversity of these Aims synergistically benefits each one.