CHAPTER 5 – Use of Short-Chain Alkynes to Locate the Nitrogenase Catalytic Site

The mechanism by which molybdenum nitrogenase catalyzes the MgATP-dependent reduction of dinitrogen to ammonia has challenged enzymologists for several years. Because the quantification of ammonia has proven to be difficult and laborious, acetylene is frequently used as an alternative substrate for probing the nitrogenase catalytic mechanism. This chapter discusses some of the problems that have denied a complete understanding of this mechanism and describes how acetylene and other short-chain alkynes have recently been used in a combination of biochemical and genetic studies. The metals and sulfurs involved in providing the acetylene-binding site are likely to be located within a particular 4Fe–4S face of the FeMo-cofactor. Nevertheless, the question of which individual atoms are specifically involved in binding, as well as the geometric features of substrate binding, remains a challenging problem. Because a more reduced state and more protons are required for dinitrogen-binding and reduction than for acetylene binding and reduction, all of the atoms that participate in dinitrogen-binding are not required for acetylene binding. The availability of altered MoFe proteins having the capacity to interact with acetylene in the resting state presents the unprecedented opportunity to determine crystallographically the structure of a MoFe protein in at least some stage of substrate binding.