Biophysical Studies of Metalloenzymes

Northwestern University, Evanston IL

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Brian Hoffman of Northwestern University is applying advanced paramagnetic resonance techniques to the solution of central problems in metallobiochemistry, the study of metal centers fundamental to life. These studies focus on multimetallic metalloenzymes that carry out life’s reactions and their synthetic analogues. One component focuses on the study of enzymes with Fe4 clusters and analogues with Fe3M, M = Fe or Mo. The focus is on ‘organometallic’ states of these clusters, which feature an Fe-C bond. Once thought to be rare in life, such states are now proposed as intermediates in terpenoid-biosynthesis enzymes and are found as intermediates central to the function of the world’s largest superfamily of metalloenzymes, the ‘radical-SAM (RS)’ enzymes, with over 700,000 members identified throughout all forms of life, which carry out a spectacular diversity of essential reactions. A second component will investigate the active site cofactor of isozymes of the enzyme nitrogenase, Fe7M, M = Mo, V, or Fe. In terms of societal Impact, the response to aims of teaching, training, and learning can be viewed as forming a pyramid. At the apex are intellectual/scientific contributions to the discipline and to the research community. Supporting these are contributions to the training of postdocs, graduate students, and undergraduates, not only in this group but in those of collaborators. A critical component of this outreach pyramid is an effort to broaden participation in the scientific enterprise, with focus on women and underrepresented minorities. Electron paramagnetic resonance (EPR)/electron-nuclear double resonance (ENDOR) studies of biomimetic synthetic [Fe3,M;S4]3+–alkyl/alkene/alkyne clusters, M = Fe, Mo, are expected to enhance understanding of intermediates of terpenoid-biosynthesis and RS enzymes, with comparison of the [Fe3,M;S4], M = Fe and Mo clusters offering insights into the role of the Mo in modulating the properties of the nitrogenase Fe7Mo catalytic cofactor. Furthermore, as shown by this program, the three nitrogenase isozymes function through a universal mechanism involving ten states, denoted En, n = 0-8. The n = even states of Mo-nitrogenase are EPR active and a majority have been characterized by EPR and ENDOR. In contrast, the n = odd states of the V- and Fe-nitrogenases are EPR-active, and given the mechanistic universality, their study will enable probing the catalytic n = odd states. New constructs of the nitrogenase isozymes will be used to probe whether reactivity differences among the three isozymes derive from influences of the different heterometals or from the differing isozyme active-site environments. In terms of broad scientific impact, better understanding of the nitrogenase enzyme is of great importance. It is well to remember that the enzyme nitrogenase carries out biological nitrogen fixation, the conversion of gaseous N2 to two molecules of ammonia, the biologically usable form of nitrogen and that approximately half the world’s human population depends on nitrogen fixation by nitrogenase. This project is supported by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences, and by the Molecular Biophysics cluster of the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.