Biophysical Studies of Metalloenzymes

Overview Biophysical Studies of Metalloenzymes; Brian M. Hoffman, Northwestern University Understanding biological nitrogen fixation, the reduction of N2 to yield two moles of ammonia, is one of the great challenges in metallobiochemistry. The current grant period marked a turning point in the understanding of nitrogenase function: our development of a mechanism for N2 reduction by this enzyme that is uniquely built on the properties of catalytic intermediates we have trapped and characterized. This period further saw breakthrough discoveries in the chemistry of biomimetic Fe complexes, both as enzyme models and as novel coordination complexes. This proposal builds on the current successes, with the first section devoted to testing, amplifying, enriching, and as needed, revising the mechanism, the second to expanding the study of biomimetic complexes, and the third to Broader Impacts. Intellectual Merit For nearly forty years after the purification of nitrogenase, nothing was known about the details of the intermediates formed during N2 reduction and the mechanism remained a mystery. Our collaborative team involving the laboratories of L. Seefeldt and D. Dean is solving this problem through their successful genetic/biochemical approaches to the isolation of catalytic intermediates in combination with this laboratory’s use of electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize them. The mechanism we proposed for N2 reduction by nitrogenase is thus uniquely built on the properties of trapped catalytic intermediates we have determined. Areas of primary focus include: (i) The nitrogenase reaction mechanism: Nitrogenase functions by successively adding six electrons and protons to N2, but we have shown that the limiting enzymological stoichiometry requires eight electrons/protons, not six. The overarching goal for the coming period is to test and develop the proposed mechanism, which incorporates this stoichiometric discrepancy as a consequence of the key mechanistic step in enzymatic activation and reduction of N2. (ii) Electronic/Geometric Structure of the Enzymatic Intermediates: Determine the structure and electronic properties of enzymatic intermediates throughout the catalytic cycle, by integrating 1,2H, 14,15N, 57Fe and 95Mo ENDOR/ESEEM spectroscopic studies with quantum chemical calculations that for the first time introduce QM/MM methods to the study of nitrogenase intermediates. (iii) The Study of Biomimetic Metal Complexes. EPR/ENDOR characterizations of biomimetic Mo and Fe complexes yield powerful constraints on the identification of substrate-derived moieties in our nitrogenase intermediates. In addition, these complexes have their own, fundamental importance to coordination chemistry as Jahn-Teller active systems that include non-classical M-H2 adducts that show novel H2 rotational dynamics and also are relevant for hydrogen catalysis and energy storage. Broader Impacts Bioavailable nitrogen derived from the reduction of N2 to two NH3 is second only to water as the limiting nutrient in plant growth. The agronomic, economic, and social significance of nitrogenase can be appreciated by recognizing that it supplies approximately one-half to two-thirds of the world’s NH3. The remaining one-third is produced by the Haber-Bosch industrial process, but this requires high temperatures and pressures and accounts for ~ 2% of the world’s total energy consumption. Whether the biological process can be more effectively exploited for human benefit i