Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide.
TL;DR: It was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH, characteristic features of the disulfide reduct enzyme class of flavoproteins.
Abstract: The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.
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TL;DR: How this very mobile and plastic suite of proteins protects host cells from this pervasive toxic metal, what roles it has in the biogeochemical cycling of Hg, and how it has been employed in ameliorating environmental contamination are the subjects of this review.
Abstract: Bacterial resistance to inorganic and organic mercury compounds (HgR) is one of the most widely observed phenotypes in eubacteria. Loci conferring HgR in Gram-positive or Gram-negative bacteria typically have at minimum a mercuric reductase enzyme (MerA) that reduces reactive ionic Hg(II) to volatile, relatively inert, monoatomic Hg(0) vapor and a membrane-bound protein (MerT) for uptake of Hg(II) arranged in an operon under control of MerR, a novel metal-responsive regulator. Many HgR loci encode an additional enzyme, MerB, that degrades organomercurials by protonolysis, and one or more additional proteins apparently involved in transport. Genes conferring HgR occur on chromosomes, plasmids, and transposons and their operon arrangements can be quite diverse, frequently involving duplications of the above noted structural genes, several of which are modular themselves. How this very mobile and plastic suite of proteins protects host cells from this pervasive toxic metal, what roles it has in the biogeochemical cycling of Hg, and how it has been employed in ameliorating environmental contamination are the subjects of this review.
941 citations
Cites background or methods from "Mercuric reductase. Purification an..."
...Low thiol titers for the proteins assayed in the original comparisons [195,196] indicate that the N-terminal cysteines were not initially reduced when the proteins were added to the assays....
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...At the same time, careful reductive titrations of the wild-type protein demonstrated that most preparations required four electrons to reach a stable EH2 state (instead of the expected two electrons) and that each two electrons gave rise to the appearance of two new cysteine thiols in the protein for a total of four [212] rather than the two previously reported [195]}....
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...Although an analogous mechanism was considered a possibility for MerA [195], chemical precedent for the interaction of thiols with Hg(II) suggested a role for high-af¢nity complexation of Hg(II) by these thiols rather than a role in its reduction....
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...Fox and Walsh [195] reported the ¢rst large-scale puri¢cation of the Tn501-encoded MerA and showed that the homodimeric enzyme exhibited spectral and biochemical properties similar to those of the pyridine nucleotide disul¢de oxidoreductase family whose most common members include glutathione reductase (GR) and lipoamide dehydrogenase....
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...Thus before proceeding with further investigation of the properties of the C-terminal segment and residues in the binding pathway, the Miller lab has recently subcloned the catalytic core region of the Tn501 MerA (residues 96^ 561) [223] in an e¡ort to obtain homogeneous protein free of the problematic proteolysis of the full-length protein [195]....
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TL;DR: Phytoremediation uses plants to remove pollutants from the environment and the recent discovery that certain chelating agents greatly facilitate metal uptake by soil-grown plants can make this technology a commercial reality in the near future.
Abstract: Phytoremediation uses plants to remove pollutants from the environment. The use of metal-accumulating plants to clean soil and water contaminated with toxic metals is the most rapidly developing component of this environmentally friendly and cost-effective technology. The recent discovery that certain chelating agents greatly facilitate metal uptake by soil-grown plants can make this technology a commercial reality in the near future.
919 citations
TL;DR: This review describes the best studied of Flavoproteins mechanisms and discusses factors possibly governing reactivity and specificity.
Abstract: Flavoproteins are a class of enzymes catalyzing a very broad spectrum of redox processes by different chemical mechanisms. This review describes the best studied of these mechanisms and discusses factors possibly governing reactivity and specificity.
549 citations
TL;DR: A set of universal procedures is brought forward, which is novel, tentative and adaptive to evaluate hyperaccumulators' feasibility before large-scale commercialization.
Abstract: Mechanism of four methods for removing hazardous heavy metal are detailed and compared-chemical/physical remediation, animal remediation, phytoremediation and microremediation with emphasis on bio-removal aspects The latter two, namely the use of plants and microbes, are preferred because of their cost-effectiveness, environmental friendliness and fewer side effects Also the obvious disadvantages of other alternatives are listed In the future the application of genetic engineering or cell engineering to create an expected and ideal species would become popular and necessary However, a concomitant and latent danger of genetic pollution is realized by a few persons To cope with this potential harm, several suggestions are put forward including choosing self-pollinated plants, creating infertile polyploid species and carefully selecting easy-controlled microbe species Bravely, the authors point out that current investigation of noncrop hyperaccumulators is of little significance in application Pragmatic development in the future should be crop hyperaccumulators (newly termed as "cropaccumulators") by transgenic or symbiotic approach Considering no effective plan has been put forward by others about concrete steps of applying a hyperaccumulator to practice, the authors bring forward a set of universal procedures, which is novel, tentative and adaptive to evaluate hyperaccumulators' feasibility before large-scale commercialization (C) 2009 Elsevier BV All rights reserved
514 citations
TL;DR: To enhance phytoremediation as a viable strategy, microbiota from the rhizosphere can play an important role, but the use of genetic engineering can also increase the success of the technique.
Abstract: Increased soil pollution with heavy metals due to various human and natural activities has led to a growing need to address environmental contamination. Some remediation technologies have been developed to treat contaminated soil, but a biology-based technology, phytoremediation, is emerging. Phytoremediation includes phytovolatilization, phytostabilization, and phytoextraction using hyperaccumulator species or a chelate-enhancement strategy. To enhance phytoremediation as a viable strategy, microbiota from the rhizosphere can play an important role, but the use of genetic engineering can also increase the success of the technique. Here we review the key information on phytoremediation, addressing both potential and limitations, resulting from the research established on this topic.
477 citations
Cites background from "Mercuric reductase. Purification an..."
...However, because of its high reactivity, Hg in the environment exists mainly as a divalent cation Hg2+; bacteria can catalyze the reduction of the mercuric ion to elemental Hg and enhance the volatilization abilities of associated plants (Fox & Walsh, 1982)....
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References
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: Some studies with glutathione reductase are reported which indicate that this enzyme does in fact possess a basically identical reaction mechanism to that of lipoyl dehydrogenase.
Abstract: Glutathione reductase has been purified from a number of sources, Escherichiu coli (l), yeast (2, 3), peas (4), rat tissues (5, 6), and erythrocytes (7). In all of the cases, the enzyme appears to be a flavoprotein, containing flavin adenine dinucleotide as prosthetic group (8). In view of the fact that the over-all reaction catalyzed by the enzyme, GSSG + TPNH + H+ & 2 GSH + TPN+, is formally so similar to that catalyzed by lipoyl dehydrogenase, another flavoprotein enzyme, oxidized lipoate (-S-S-) + DPNH + H+ F! reduced lipoate ((-SH),) + DPN+, it is of obvious interest to determine whether the two enzymes have similar reaction mechanisms. In the case of pig heart lipoyl dehydrogenase, it has been found that a protein disulfide linkage (9-11) is concerned in catalysis, in close concert with the FAD prosthetic group. The anaerobic addition of reducing substrate (either DPNH or dihydrolipoyl derivatives) results in the formation of a stable 2-electron reduction intermediate which has been shown to be extremely important in catalysis (12). This intermediate appears to be a complex between the enzyme flavin semiquinone and a sulfur radical formed from the active center disulfide (13). The intermediate has a very characteristic absorption spectrum, quite different from that found with half-reduced forms of other flavoproteins. Our interest in the possible similarity of lipoyl dehydrogenase and glutathione reductase was considerably aroused by the published spectra of yeast glutathione reductase (3) which showed characteristics very similar to those found with lipoyl dehydrogenase. It was suggested therefore that the two enzymes may have very similar reaction mechanisms (14), i.e. that in glutathione reductase a protein disulfide active center might also be operative. This suggestion appeared to have been discounted by later results of Colman and Black (15) who showed by experiments with W-labeled N-ethylmaleimide that only 1 sulfhydryl group was liberated on treatment of the enzyme with TPNH. In this paper, we wish to report on some studies with glutathione reductase which indicate that this enzyme does in fact possess a basically identical reaction mechanism to that of lipoyl dehydrogenase
453 citations
TL;DR: The mercury cycle in the biosphere and biological methylation of mercury and microbial resistance to mercury and organomercurials are studied.
Abstract: BIOTRANSFORMA nONS OF TOXIC MET AL CAnONS . Mercury . The mercury cycle in the biosphere .. Biological methylation of mercury . Microbial resistance to mercury and organomercurials .
413 citations
375 citations
Book•
27 Apr 1977
TL;DR: Ten basic techniques in contemporary biochemical and molecular biological studies are dealt with and the theoretical aspect of each technique is examined and a section for its use in a carefully controlled experiment is provided.
Abstract: Deals with ten basic techniques in contemporary biochemical and molecular biological studies. Examines the theoretical aspect of each technique and provides a section for its use in a carefully controlled experiment. Experiments, written in computer program format, conclude with the data that should be obtained if experiments are performed properly.
330 citations