Trace metal requirements for microbial enzymes involved in the production and consumption of methane and nitrous oxide

Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO_2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH_4), and nitrous oxide (N_2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH_4 and N_2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH_4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N_2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N_2O reductase, the only known enzyme capable of microbial N_2O conversion to N_2, have only been found in classical denitrifiers. Accumulation of N_2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N_2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide-metal scavenging

Oxygen Activation by Mononuclear Mn, Co, and Ni Centers in Biology and Synthetic Complexes

The active sites of metalloenzymes that catalyze O2-dependent reactions generally contain iron or copper ions. However, several enzymes are capable of activating O2 at manganese or nickel centers instead, and a handful of dioxygenases exhibit activity when substituted with cobalt. This minireview summarizes the catalytic properties of oxygenases and oxidases with mononuclear Mn, Co, or Ni active sites, including oxalate-degrading oxidases, catechol dioxygenases, and quercetin dioxygenase. In addition, recent developments in the O2 reactivity of synthetic Mn, Co, or Ni complexes are described, with an emphasis on the nature of reactive intermediates featuring superoxo-, peroxo-, or oxo-ligands. Collectively, the biochemical and synthetic studies discussed herein reveal the possibilities and limitations of O2 activation at these three “overlooked” metals

Structure and Mechanisms of Metalloenzymes for Incorporation of Water Across CN and C-Cl Bonds

Metalloenzymes are widely involved in water incorporation across CN and C-Cl bonds.1 In this research, the structure and mechanisms of two novel metalloenzymes that incorporate water across CN and C-Cl bonds were examined by biophysical approaches, computational modeling, and crystallographic methods.In the research of water incorporation across CN bonds, a novel eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase) was characterized, and the functional role of a (His)17 section and an insert region in the MbNHase, were examined by gene modifications. Each of these MbNHase enzymes provided an 22 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions The kinetic studies and metal analysis establish that neither the (His)17 nor the entire insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.In the research of water incorporation across C-Cl bonds, a novel Zn(II)-dependent chlorothalonil hydrolytic dehalogenase from Pseudomonas sp. CTN-3 (Chd) was characterized and analyzed by a new direct spectrophotometric assay, metal analysis, and crystallography methods. A single Zn(II) ions was found to bind per Chd monomer. Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0 while multiple turnover pre-steady state stopped-flow data suggested a three-step model. The combination of these data along with pH dependent studies, allowed a catalytic mechanism for Chd to be proposed for the first time. The X-ray crystal structure of a fifteen residue N-terminal truncated form of the Chd (ChdT) was solved using single wavelength anomalous dispersion (SAD) to a resolution of 1.96 Å in the primitive orthorhombic space group P212121. ChdT is the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT is a “head-to-tail” homodimer, formed between two -helices from each monomer, with three Zn(II) binding sites, one of which is the active site Zn(II) while the third forms a structural site at the homodimer interface. The structural Zn(II) ion is not accessible to the bulk solvent. The active site Zn(II) ion resides in a slightly distorted trigonal bipyramid or TBP geometry with His117, His257, Asp116, Asn216, and water/hydroxide as ligands. A conserved His residue (His114) is hydrogen bound to the Zn(II) bound water/hydroxide and likely functions as the general acid/base. Substrate binding was examined by docking TPN into the hydrophobic channel with the most energetically favorable pose occurring for a TPN orientation that coordinates to the active site Zn(II) ions via a CN and that maximizes a π-π interaction with Trp227. The combination of the structure and substrate docking studies with the previously reported kinetic studies, has allowed a refined catalytic mechanism to be propose

Artificial Metalloenzymes

In de natuur wordt een groot deel van de chemische transformaties uitgevoerd door complexe moleculen, die bekend staan als enzymen. Deze enzymen zijn katalysatoren die vaak bouwstenen produceren van één spiegelbeeld. Het produceren van bouwstenen van slechts een spiegelbeeld is belangrijk voor het maken van bijvoorbeeld medicijnen. Echter natuurlijke enzymen kunnen slechts een gelimiteerde set van deze transformaties uitvoeren. Homogene katalyse, vaak gebruikt in organische chemie, kan een veel bredere set aan transformatie uitvoeren, maar niet altijd van één spiegelbeeld. Artificiële metaalenzymen maken gebruik van de voordelen van de twee eerder genoemde katalysatoren en combineert deze tot een. Zo’n katalysator bestaat uit een biomolecuul en een metaalcomplex. Toepassing van deze katalysatoren is al zeer succesvol gebleken, echter het aantal biomoleculen dat beschikbaar is voor de constructie van artificiële metaalenzymen is beperkt.Dit proefschrift beschrijft een nieuwe benadering voor ontwikkeling en constructie van artificiële metaalenzymen.Een actieve centrum, waar de chemische transformatie plaats vindt, werd gecreëerd op een interface van een eiwit dat bestaat uit twee gelijk delen. Hiervoor werd het eiwit LmrR gebruikt. Twee benaderingen om het metaal complex te verankeren aan LmrR werden gedemonstreerd, namelijk op een covalente en niet covalente manier. De artificiële metaalenzymen die hieruit ontstonden, konden verschillende katalytische transformaties uitvoeren waarbij zeer goede resultaten werden behaald. Dat wil zeggen dat een hoog percentage van een spiegelbeeld werden gevormd. Een verdere studie van deze katalysatoren gaf een beter inzicht hoe deze transformaties uitgevoerd werden.In nature, a large scope of chemical transformations are performed by complex molecules, known as enzymes. Enzymes are catalysts that are able to produce building blocks of just one mirror image. The production of these building blocks possessing just one mirror image is important for the construction of medicine, for example. However, enzymes have a very limited scope of transformation they can achieve. On the other hand, homogenous catalysis, can perform a much wider scope of transformations. However, it does not always produce one mirror image of the building block. Artificial metalloenzymes utilizes the benefits of the catalysts mentioned previously. An artificial metalloenzymes consists of a metal complex and a biomolecule. Artificial metalloenzymes proved to be very successful, however the number of available biomolecules for the construction of artificial metalloenzymes is limited.This thesis describes a new approach for the development and construction of artificial metalloenzymes.An active site, in which the chemical transformation takes place, was constructed on the interface of a protein that consisted of two equal parts. The protein that was used was LmrR. Different approaches to anchor the metal complex covalent or non-covalent are described in this thesis. The resulting artificial metalloenzymes were able to catalyze a variety of chemical transformations with very good results, i.e. a high percentage of one of the two mirror images of the products were formed. A study of these artificial metalloenzyme gave more insight in how these transformations were performed