Exploring new frontiers of nitrogenase structure and mechanism John W Peters and Robert K Szilagyi The mechanism of the complex enzyme nitrogenase has long been one of the most challenging problems in bioinorganic chemistry. The complexity of the metal centers of nitrogenase has stretched the boundaries of biochemical, physical and computational tools for providing insights into its structure and chemical function. Recently, there have been several key advances in crystallography and spectroscopy that have impacted the way the nitrogenase mechanism is approached. These advances have opened new frontiers in nitrogenase research, which has started to reveal novel details about the molecular structure, substrate binding and reduction. Here, we discuss these recent advances and their implications on the future of nitrogenase research. Addresses Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, 59717, USA Corresponding author: Peters, John W (john.peters@chemistry.montana.edu) Current Opinion in Chemical Biology 2006, 10:101–108 This review comes from a themed issue on Bioinorganic chemistry Edited by David M Dooley and Amy Rosenzweig Available online 28th February 2006 1367-5931/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.02.019 Introduction Nitrogen fixation has always been considered of funda- mental importance, not only for its significance in global nutrition, but also because of the relevance of nitrogenase as a model system for examining processes such as multi- ple electron oxidation reduction reactions, complex bio- logical metal assembly, and even nucleotide-dependent signal transduction. Nitrogenase occurs in molybdenum, vanadium and iron forms, with unique metal centers located at the sites of nitrogen binding and reduction. Molybdenum nitrogenase is by far the most extensively characterized and contains two components termed the Fe protein and MoFe protein. The Fe protein is a 60 kD homodimer that contains a single [4Fe–4S] cubane and functions in MgATP hydrolysis and electron transfer to the substrate reduction component, the 230 kD MoFe protein heterotetramer. The MoFe-pro- tein contains two types of metal clusters: the P clusters and the FeMo-cofactors (FeMo-cos) (Figure 1). The structures of the Fe and MoFe proteins have been deter- mined, as well as several stabilized nitrogenase Fe pro- tein–MoFe protein complexes [1,2  ]. Several interesting aspects of nitrogenase structure and mechanism are being actively researched, including the biosynthesis of the nitrogenase and insertion of the FeMo-co [3–5], the specific role of the nitrogenase P cluster [6], the role of MgATP in nitrogenase catalysis [2  ,7  ], the mode and manner of substrate binding [8  ,9  ], and the nitrogenase mechanism. The current review focuses on a subset of these topics and highlights the progress concerning the role of MgATP in nitrogenase catalysis and the recent experimental and computational studies that have pro- vided significant mechanistic insights into substrate reduction at the FeMo-co. The role of MgATP in nitrogenase catalysis An important issue concerning nitrogenase catalysis is the role of MgATP binding and hydrolysis, which is required for nitrogen reduction unlike other multiple-electron reduction reactions in biochemistry. Each individual elec- tron transfer reaction between the nitrogenase compo- nents requires the binding and hydrolysis of at least two MgATP molecules [10]. Although the role of MgATP binding and hydrolysis in the process is yet to be unequi- vocally determined, it is a rational assumption that this requirement is part of a gating mechanism that effectively maintains unidirectional electron transfer toward sub- strate reduction even as multiple electrons accumulate on the MoFe protein, as reduced metal clusters or sub- strate reduction intermediates. A site-directed variant of the Azotobacter vinelandii Fe protein has been characterized, which has a single amino acid deletion (Leu127D) in a nucleotide dependent ‘Switch’ region that directly links the nucleotide binding site to the [4Fe–4S] cluster [11]. The Leu127D variant of the Fe protein has been extensively studied by biochem- ical and physical methods and has been shown to behave similarly, in many respects, to the MgATP-bound state of the native Fe protein. Although this variant does not support nitrogen reduction and catalyzes very low rates of MgATP hydrolysis, it is capable of forming a stable complex with the MoFe protein in the absence of added nucleotides [12] that closely resembles the native Fe protein–MoFe protein complex stabilized in the presence of MgADP and tetrafluoroaluminate [13,14](Figure 1). The structural characterization of the Leu127D Fe pro- tein variant revealed a large degree of rigid body reor- ientation of the subunits relative to the previously reported structures of Fe protein [7  ]. During nitrogen- ase catalysis, an analogous rotation of the subunits away www.sciencedirect.com Current Opinion in Chemical Biology 2006, 10:101–108