Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

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Structural and Functional Aspects of Metal Sites in Biology

Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidation-reduction reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addition to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidation states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biological prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity of function.

http://science.sciencemag.org/content/sci/277/5326/653.full.pdf

Iron-Sulfur Clusters: Nature's Modular, Multipurpose Structures

Molybdenum is the only second-row transition metal required by most living organisms, and is nearly universally distributed in biology. Enzymes containing molybdenum in their active sites have long been recognized,1 and at present over 50 molybdenum-containing enzymes have been purified and biochemically characterized; a great many more gene products have been annotated as putative molybdenum-containing proteins on the basis of genomic and bioinformatic analysis.2 In certain cases, our understanding of the relationship between enzyme structure and function is such that we can speak with confidence as to the detailed nature of the reaction mechanism and, with the availability of high-resolution X-ray crystal structures, the specific means by which transition states are stabilized and reaction rate is accelerated within the friendly confines of the active site. At the same time, our understanding of the biosynthesis of the organic cofactor that accompanies molybdenum (variously called molybdopterin or pyranopterin), the manner in which molybdenum is incorporated into it, and then further modified as necessary prior to insertion into apoprotein has also (in at least some cases) become increasingly well understood.

It is now well-established that all molybdenum-containing enzymes other than nitrogenase (in which molybdenum is incorporated into a [MoFe7S9] cluster of the active site) fall into three large and mutually exclusive families, as exemplified by the enzymes xanthine oxidase, sulfite oxidase, and DMSO reductase; these enzymes represent the focus of the present account.3 The structures of the three canonical molybdenum centers in their oxidized Mo(VI) states are shown in Figure 1, along with that for the pyranopterin cofactor. The active sites of members of the xanthine oxidase family have an LMoVIOS-(OH) structure with a square-pyramidal coordination geometry. The apical ligand is a Mo=O ligand, and the equatorial plane has two sulfurs from the enedithiolate side chain of the pyranopterin cofactor, a catalytically labile Mo–OH group, and most frequently a Mo=S. Nonfunctional forms of these enzymes exist in which the equatorial Mo=S is replaced with a second Mo=O; in at least one member of the family the Mo=S is replaced by a Mo=Se, and in others it is replaced by a more complex –S–Cu–S–Cys to give a binuclear center. Members of the sulfite oxidase family have a related LMoVIO2(S–Cys) active site, again square-pyramidal with an apical Mo=O and a bidentate enedithiolate Ligand (L) in the equatorial plane but with a second equatorial Mo=O (rather than Mo–OH) and a cysteine ligand contributed by the protein (rather than a Mo=S) completing the molybdenum coordination sphere. The final family is the most diverse structurally, although all members possess two (rather than just one) equiv of the pyranopterin cofactor and have an L2MoVIY(X) trigonal prismatic coordination geometry. DMSO reductase itself has a catalytically labile Mo=O as Y and a serinate ligand as X completing the metal coordination sphere of oxidized enzyme. Other family members have cysteine (the bacterial Nap periplasmic nitrate reductases), selenocysteine (formate dehydrogenase H), –OH (arsenite oxidase), or aspartate (the NarGHI dissimilatory nitrate reductases) in place of the serine. Some enzymes have S or even Se in place of the Mo=O group. Members of the DMSO reductase family exhibit a general structural homology to members of the aldehyde:ferredoxin oxidoreductase family of tungsten-containing enzymes;4 indeed, the first pyranopterin-containing enzyme to be crystallographically characterized was the tungsten-containing aldehyde:ferredoxin oxidoreductase from Pyrococcus furiosus,5 a fact accounting for why many workers in the field prefer “pyranopterin” (or, perhaps waggishly, “tungstopterin”) to “molybdopterin”. The term pyranopterin will generally be used in the present account.




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Figure 1


Active site structures for the three families of mononuclear molybdenum enzymes. The structures shown are, from left to right, for xanthine oxidase, sulfite oxidase, and DMSO reductase. The structure of the pyranopterin cofactor common to all of these enzymes (as well as the tungsten-containing enzymes) is given at the bottom.

/pdf/the-mononuclear-molybdenum-enzymes-huemv59go8.pdf

The Mononuclear Molybdenum Enzymes

The authors review the development of extended x-ray absorption fine structure (EXAFS) within the last decade. Advances in experimental techniques have been largely stimulated by the availability of synchrotron radiation. The theory of EXAFS has also matured to the point where quantitative comparison with experiments can be made. The authors review in some detail the analysis of EXAFS data, starting from the treatment of raw data to the extraction of distances and amplitude information, and they also discuss selected examples of applications of EXAFS chosen to illustrate both the strength and limitations of EXAFS as a structural tool.

Extended x-ray absorption fine structure—its strengths and limitations as a structural tool

One of the two protein components of the nitrogenase enzyme system (the so-called MoFe protein) contains two Mo atoms and 24 to 32 Fe atoms per 220 to 270,000 molecular weight. Despite many hypotheses about the Mo site and its involvement in dinitrogen reduction, there has been no spectroscopic means of unambiguously probing the state of Mo. In this paper, the results of x-ray absorption spectroscopy studies are described and the analysis of these results provides the first direct evidence of the Mo coordination environment in the MoFe protein from Clostridium pasteurianum nitrogenase. The Mo is found to have primarily S ligation and in the resting state (''semireduced form'') of the protein there appears to be no Mo=O species present. On air oxidation, however, clear evidence is found for the presence of Mo=O. The interaction of the Mo with one other metal, most probably Fe and most certainly not Mo, is also seen and it is suggested that the Mo is present in a Mo--Fe--S cluster not yet adequately modeled by any synthetic inorganic system. 8 figures, 6 tables, 40 references.

/pdf/the-molybdenum-site-of-nitrogenase-preliminary-structural-1ytucutp1j.pdf

The molybdenum site of nitrogenase. Preliminary structural evidence from x-ray absorption spectroscopy

The molybdenum site of nitrogenase. 2. A comparative study of molybdenum-iron proteins and the iron-molybdenum cofactor by x-ray absorption spectroscopy

Quantitative extrusions of the Fe4S4 cores of the active sites of ferredoxins and the hydrogenase of Clostridium pasteurianum.

Letter: Molybdenum X-ray absorption edge spectra. The chemical state of molybdenum in nitrogenase.

Nitrogenase is a complex bacterial enzyme system that is responsible for the conversion of atmospheric N/sub 2/ to ammonia. The structure and function of molybdenum in the MoFe protein of this system has been the subject of a number of investigations, including the use of X-ray absorption spectroscopy. This paper reports the results of the authors recent studies on several states of the MoFe protein and its FeMo cofactor (which is extruded by treatment with N-methylformamide). Mo K-edge (XANES) and extended fine structure (EXAFS) spectra have been recorded to high energies above the absorption edge with excellent signal-to-nose on the semireduced form of the MoFe protein from both Clostridium pasteurianum and Azotobacter vinelandii and on the as isolated FeMo-co and FeMo-co treated with benzenethiol and with benzeneselenol. In all of the states studied, EXAFS results reveal that the Mo is in an environment that contains two or three oxygen (or nitrogen) atoms at 2.10-2.12 A, three to five S atoms at 2.37 A, and three to four Fe atoms at 2.68-2.70 A. The numbers of these ligands change upon removal of the cofactor from the protein as discussed in the paper. For FeMo-co, comparisons also show that thil/selenol is notmore » binding directly to the Mo site. The results of these EXAFS (and their XANES published earlier) definitely show the presence of several low-Z ligands and are not compatible with a tetrahedral arrangement of only nearest S neighbors at the Mo site.« less

L. E. Mortenson

Papers

The molybdenum site of nitrogenase. Preliminary structural evidence from x-ray absorption spectroscopy

The molybdenum site of nitrogenase. 2. A comparative study of molybdenum-iron proteins and the iron-molybdenum cofactor by x-ray absorption spectroscopy

Quantitative extrusions of the Fe4S4 cores of the active sites of ferredoxins and the hydrogenase of Clostridium pasteurianum.

Letter: Molybdenum X-ray absorption edge spectra. The chemical state of molybdenum in nitrogenase.

Structural studies of the molybdenum site in the MoFe protein and its FeMo cofactor by EXAFS