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Investigating the roles of protein-protein interactions and conformational changes in nitrogenase catalysis

  • Author(s): Katz, Faith
  • Advisor(s): Tezcan, Faik A.
  • et al.
Abstract

Many adverse environmental effects are associated with the industrial production of commodity chemicals like ammonia (NH3) from atmospheric dinitrogen (N2). Nitrogenase is the only enzyme capable of reducing N2 gas to a bioavailable form. Remarkably, the two-component protein system nitrogenase achieves this reaction at room temperature and atmospheric pressure, using ATP as the energy source. Though biological nitrogen fixation has been studied for nearly a century, the precise mechanism by which Nature activates N2 remains elusive. The overarching goal of this dissertation is to understand the molecular mechanism of biological nitrogen fixation, as this will lead to the development of more environmentally sustainable catalysts for industrial N2 activation and accelerate efforts to engineer the ability to fix nitrogen into plants.

Specifically, protein-protein interactions between and conformational changes within the nitrogenase component proteins are investigated in this work to gain insight as to why biological nitrogen fixation requires a specific, ATP-dependent electron donor protein to support catalysis. Through the study of site-directed nitrogenase mutants, a negatively-charged patch on the surface of the electron donor protein and a positively-charged patch on the surface of the catalytic component protein of nitrogenase were found to associate during turnover en route to the formation of a complex capable of interprotein electron transfer. While changes in the abilities of these mutant proteins to reduce H+ to H2 and C2H2 to C2H4 were explained well by the consensus Thorneley-Lowe model for nitrogenase reactivity, the model could neither predict nor explain the behavior of either wild-type or mutant proteins under an N2 atmosphere, performing the biologically relevant reduction of N2 to yield NH3 and H2.

Finally, the possibility for the electron donor protein in nitrogenase to transduce structural changes within the catalytic protein component upon binding is evaluated, and a mechanism for conformational gating of electron transfer in nitrogenase proposed. The findings of this dissertation would not have been achieved without significant efforts to create, optimize, and improve experimental methods in molecular biology and analytical biochemistry related to the study of nitrogenase, which are the subjects of the final chapters in this dissertation.

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