Electron transfer reactions and the role of MgATP hydrolysis in nitrogenase catalysis

The conversion of atmospheric dinitrogen to ammonia is a major life-supporting process on this planet. For the past century, approximately one fourth of the fixed nitrogen demand on this planet has been provided by the Haber-Bosch process for producing nitrogenous fertilizer. Three fourths of the fixed nitrogen demand has been provided by biological nitrogen fixation. Presently, the best characterized biological nitrogen fixing system, or nitrogenase, is a two-component metalloenzyme that catalyzes the reduction of nitrogen with the overall reaction usually depicted in the following way: N2 + 8H+ + 8e-- + 16MgATP → 2NH3 + H2 + 16MgADP + 16Pi (Eqn. I). It is clear why protons and electrons are required in this process, but the role of MgATP hydrolysis has remained a central question in the nitrogenase mechanism for over 40 years. This dissertation focused on the role of MgATP in electron transfer and the catalytic mechanism of nitrogenase.; The two components of this system are the iron (Fe) protein and the molybdenum-iron (MoFe) protein. These names are primarily derived from the metal composition of the respective proteins. What we know about these two proteins and their respective roles in nitrogenase catalysis is discussed in detail in Chapter II. In general, it can be said that the Fe protein serves as the reductant and molecular switch that couples unidirectional electron flow in the nitrogenase complex to events associated with MgATP binding and hydrolysis. In the catalytic cycle of nitrogenase, the reduced Fe protein binds two molecules of MgATP and forms a protein-protein complex with the MoFe protein. Subsequent electron transfer from the Fe protein to the MoFe protein is coupled to the hydrolysis of MgATP. The work compiled in this dissertation examined nucleotide interaction, signal transduction, and electron transfer and can be summarized as follows.; The thermodynamics of nucleotide interaction with the Fe protein is examined by the use of isothermal titration calorimetry (Chapter III). This technique has the advantage of being able to establish for the first time the thermodynamics of nucleotide interaction at each of two binding sites on the Fe protein. Site-directed mutagenesis of residues in the nucleotide binding site of the Fe protein was performed and provided substantial insight into the mechanism of MgATP hydrolysis and signal transduction pathways in the Fe protein (Chapter IV and Chapter VI). This information allowed many aspects of the relationship between MgATP hydrolysis and electron transfer to be investigated (Chapters VII to XI). In addition to the work with Fe protein, it is demonstrated in Chapter X of this dissertation that electron transfer is coupled to proton transfer for the p2+/1+ redox couple.; The observations presented in this dissertation are considered in the context of our present knowledge of nitrogenase and are used to develop a model of the nitrogenase mechanism. In particular, this work has provided insight into many fundamental questions concerning electron transfer and the role of MgATP in the nitrogenase mechanism.