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

Natural organic biomass burning creates black carbon which forms a considerable proportion of the soil’s organic carbon. Due to black carbon’s aromatic structure it is recalcitrant and has the potential for long-term carbon sequestration in soil. Soils within the Amazon-basin contain numerous sites where the ‘dark earth of the Indians’ (Terra preta de Indio, or Amazonian Dark Earths (ADE)) exist and are composed of variable quantities of highly stable organic black carbon waste (‘biochar’). The apparent high agronomic fertility of these sites, relative to tropical soils in general, has attracted interest. Biochars can be produced by ‘baking’ organic matter under low oxygen (‘pyrolysis’). The quantities of key mineral elements within these biochars can be directly related to the levels of these components in the feedstock prior to burning. Their incorporation in soils influences soil structure, texture, porosity, particle size distribution and density. The molecular structure of biochars shows a high degree of chemical and microbial stability. A key physical feature of most biochars is their highly porous structure and large surface area. This structure can provide refugia for beneficial soil micro-organisms such as mycorrhizae and bacteria, and influences the binding of important nutritive cations and anions. This binding can enhance the availability of macro-nutrients such as N and P. Other biochar soil changes include alkalisation of soil pH and increases in electrical conductivity (EC) and cation exchange capacity (CEC). Ammonium leaching has been shown to be reduced, along with N2O soil emissions. There may also be reductions in soil mechanical impedance. Terra preta soils contain a higher number of ‘operational taxonomic units’ and have highly distinctive microbial communities relative to neighbouring soils. The potential importance of biochar soil incorporation on mycorrhizal fungi has also been noted with biochar providing a physical niche devoid of fungal grazers. Improvements in soil field capacity have been recorded upon biochar additions. Evidence shows that bioavailability and plant uptake of key nutrients increases in response to biochar application, particularly when in the presence of added nutrients. Depending on the quantity of biochar added to soil significant improvements in plant productivity have been achieved, but these reports derive predominantly from studies in the tropics. As yet there is limited critical analysis of possible agricultural impacts of biochar application in temperate regions, nor on the likelihood of utilising such soils as long-term sites for carbon sequestration. This review aims to determine the extent to which inferences of experience mostly from tropical regions could be extrapolated to temperate soils and to suggest areas requiring study.

Potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

Insect guts present distinctive environments for microbial colonization, and bacteria in the gut potentially provide many beneficial services to their hosts. Insects display a wide range in degree of dependence on gut bacteria for basic functions. Most insect guts contain relatively few microbial species as compared to mammalian guts, but some insects harbor large gut communities of specialized bacteria. Others are colonized only opportunistically and sparsely by bacteria common in other environments. Insect digestive tracts vary extensively in morphology and physicochemical properties, factors that greatly influence microbial community structure. One obstacle to the evolution of intimate associations with gut microorganisms is the lack of dependable transmission routes between host individuals. Here, social insects, such as termites, ants, and bees, are exceptions: social interactions provide opportunities for transfer of gut bacteria, and some of the most distinctive and consistent gut communities, with specialized beneficial functions in nutrition and protection, have been found in social insect species. Still, gut bacteria of other insects have also been shown to contribute to nutrition, protection from parasites and pathogens, modulation of immune responses, and communication. The extent of these roles is still unclear and awaits further studies.

The gut microbiota of insects - diversity in structure and function.

Biological N2 fixation represents the major source of N input in agricultural soils including those in arid regions. The major N2-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N2-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N2-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.

Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.

https://www.pnas.org/content/pnas/74/8/3249.full.pdf

Isolation of an iron-molybdenum cofactor from nitrogenase

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ. Images

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

SOME termites can live on a diet of cellulose filter paper1. This characteristic prompted Hungate2 to question the possibility of N2 fixation in termites by estimating the N balance of growing termite colonies, but he found no net increase in total colony N. Toth3, on the other hand, incubated macerated termites in an N-free organic medium and observed an increase in fixed N in the solution. He used long incubation times, however, and possibly selected for N2-fixing organisms in the medium. We decided to reinvestigate the question of N2 fixation in termites by exploiting the sensitivity of the acetylene reduction assay, a reliable indicator of N2 fixation4,5. Preliminary reports of these results have already been published6,7.

Nitrogen Fixation in Termites

Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence.

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

Winston J. Brill

Papers

Isolation of an iron-molybdenum cofactor from nitrogenase

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Nitrogen Fixation in Termites

Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence.

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