James B. Howard

Structural basis of biological nitrogen fixation

Biological nitrogen fixation is mediated by the nitrogenase enzyme system that catalyses the ATP dependent reduction of atmospheric dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the MoFe-protein with the FeMo-cofactor that provides the active site for substrate reduction, and the Fe-protein that couples ATP hydrolysis to electron transfer. An overview of the nitrogenase system is presented that emphasizes the structural organization of the proteins and associated metalloclusters that have the remarkable ability to catalyse nitrogen fixation under ambient conditions. Although the mechanism of ammonia formation by nitrogenase remains enigmatic, mechanistic inferences motivated by recent developments in the areas of nitrogenase biochemistry, spectroscopy, model chemistry and computational studies are discussed within this structural framework.

Nitrogenase: Standing at the Crossroads

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, which is central to the process of biological nitrogen fixation. Recent progress towards establishing the mechanism of action of this complex metalloenzyme reflects the contributions of a combination of structural, biochemical, spectroscopic, synthetic and theoretical approaches to a challenging problem with implications for a range of biochemical and chemical systems.

How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation

During the process of biological nitrogen fixation, the enzyme nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein; the Fe protein mediates the coupling of ATP hydrolysis to interprotein electron transfer, whereas the active site of the MoFe protein contains the polynuclear FeMo cofactor, a species composed of seven iron atoms, one molybdenum atom, nine sulfur atoms, an interstitial light atom, and one homocitrate molecule. This Perspective provides an overview of biological nitrogen fixation and introduces three contributions to this special feature that address central aspects of the mechanism and assembly of nitrogenase.

Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase.

Making nitrogen available for biosynthesis Nitrogen gas (N2) is abundant in Earths atmosphere; however, it must be converted into a bioavailable form before it can be incorporated into biomolecules. The enzyme nitrogenase, which is made up of two metalloproteins, converts N2 into bioavailable ammonia. One of these, the MoFe-protein, contains a complex metal center, the FeMo cofactor, where the triple N2 bond is reduced. Understanding how nitrogenase achieves the reduction of N2 has been a long-term goal. Spatzal et al. present the structure of MoFe-protein bound to carbon monoxide (see the Perspective by Hogbom). Although this is an inhibitor rather than the natural substrate, the structure gives insight into how the FeMo metallocluster rearranges to achieve substrate reduction. Science, this issue p. 1620 The structure of an inhibitor bound to nitrogenase reveals rearrangements in the active-site metallocluster. [Also see Perspective by Hognom] The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO)–inhibited nitrogenase molybdenum-iron (MoFe)–protein at 1.50 angstrom resolution, which reveals a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The μ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 angstrom resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase.

Perspectives on Non-Heme Iron Protein Chemistry

Publisher Summary Non-heme iron proteins contain a magnificent assortment of iron sites having a multitude of chemical and structural properties. The catalog of iron centers is like the taxonomy of insects, a seemingly limitless variation of a few structural themes, yet each new form sufficiently different to define a new species. This chapter provides an overview of the major classes with an emphasis on proteins, for which a crystal structure is available— namely, mononuclear iron proteins, binuclear octahedral iron proteins, and tetrahedral iron Fe: S proteins. The chapter reviews the types of protein iron structures are surveyed and a discussion of some methods and problems associated with establishing the iron center type. The current status and recent developments for a limited number of proteins from the major iron classes are focused. The diversity of iron center structures and functions are emphasized. A summary of the protein crystallographic structures of non-heme iron centers and ligands is tabulated. Two types of iron prosthetic groups are classified by spectroscopic methods as having nominal octahedral coordination. The hypothesis of what protein structural constraints impose a specific type of Fe: S cluster can be tested by a combination of mutagenesis, spectroscopy, and crystallography of smaller Fe: S proteins.

On the role of γ-carboxyglutamic acid in calcium and phospholipid binding

Summary The vitamin K-dependent amino acid, γ-carboxyglutamic acid, is essential for calcium binding by prothrombin and blood clotting factor X. The studies reported here demonstrate that while γ-carboxyglutamic acid-containing peptides will bind calcium, a secondary/tertiary protein structure is also necessary to form the tight calcium binding sites which are required for binding these proteins to phospholipid surfaces.

Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001

Structural Evidence for Asymmetrical Nucleotide Interactions in Nitrogenase

The roles of ATP hydrolysis in electron-transfer (ET) reactions of the nitrogenase catalytic cycle remain obscure. Here, we present a new structure of a nitrogenase complex crystallized with MgADP and MgAMPPCP, an ATP analogue. In this structure the two nucleotides are bound asymmetrically by the Fe-protein subunits connected to the two different MoFe-protein subunits. This binding mode suggests that ATP hydrolysis and phosphate release may proceed by a stepwise mechanism. Through the associated Fe-protein conformational changes, a stepwise mechanism is anticipated to prolong the lifetime of the Fe-protein-MoFe-protein complex and, in turn, could orchestrate the sequence of intracomplex ET required for substrate reduction.

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

Abstract In 80% dimethyl sulfoxide/H2O, Azotobacter ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the Azotobacter ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.

Properties of A Ca2+ binding peptide from prothrombin

Summary The peptide containing the vitamin K-dependent Ca2+ binding region of prothrombin was isolated as described by Nelsestuen, G.L., and Suttie, J. ( Proc . Natl . Acad . Sci . (USA) (1973) 70 , 3366–3370) and its amino acid composition reported. From the acid-base titration of the peptide, a large excess of free carboxyl groups was revealed. Quantitation of the carboxyl groups indicated eight additional carboxyls which are probably a component of the unknown prosthetic group(s) attached to the peptide. The carboxyl groups of the peptide were shown to be required for quantitative adsorption onto barium citrate and presumably for binding of Ca2+.