Enzymatic Detoxification of Cyanide: Clues from Pseudomonas aeruginosa Rhodanese

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J Mol Microbiol Biotechnol 2008;15:199–211 DOI: 10.1159/000121331

Published online: July 28, 2008

Enzymatic Detoxification of Cyanide: Clues from Pseudomonas aeruginosa Rhodanese Rita Cipollone a Paolo Ascenzi a, b Paola Tomao c Francesco Imperi a Paolo Visca a, b a

Dipartimento di Biologia, Università ‘Roma Tre’; b Istituto Nazionale per le Malattie Infettive IRCCS ‘Lazzaro Spallanzani’, Roma, and c Dipartimento di Medicina del Lavoro, Istituto Superiore per la Prevenzione e la Sicurezza sul Lavoro, Monteporzio Catone (Roma), Italy

Key Words Biocatalysis ⴢ Bioremediation ⴢ Cyanide ⴢ Sulfurtransferase ⴢ Pseudomonas aeruginosa rhodanese

transferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.

Abstract Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfur-

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Introduction

The negatively charged, nucleophilic cyanide ion, CN–, consists of one carbon atom triple-bonded to one nitrogen atom with the negative charge that primarily rests on the carbon atom. When cyanide is bound to the hydrogen ion (H+), it is a gas referred to as hydrocyanic acid or hydrogen cyanide (HCN). Hydrogen cyanide has pKa of 9.21 at 25 ° C. Therefore, cyanide is largely present as volatile HCN at physiological pH values, and the term cyanide will be used hereafter to indicate both HCN and CN–. Cyanide is a dreaded compound because of its toxic properties. It causes inhibition of cellular growth by forming stable complexes with key metabolic enzymes or their substrates. Three main inhibitory mechanisms are known to date: (1) reaction with keto-compounds to give Paolo Visca Dipartimento di Biologia Universita Roma Tre, Viale G. Marconi 446 IT–00146 Roma (Italy) Tel. +39 06 5733 6347, Fax +39 06 5733 6321, E-Mail [email protected]

cyanohydrin derivatives, (2) reaction with Schiff-base intermediates to form very stable nitrile derivatives, and (3) chelation of di- and trivalent metals in metalloenzymes. A typical case of metabolic inhibition is the irreversible binding of cyanide to cytochrome c oxidase which results in the blockage of the aerobic cellular respiration [Solomonson, 1981]. Cyanide binds to the ferric heme iron of the enzyme with a 1:1 stoichiometry. Consequently, cytochrome c oxidase becomes unable to catalyze the reactions in which electrons would be transferred from the reduced cytochrome c to oxygen. Therefore, oxygen utilization is impaired, with resultant reduction or cessation of aerobic metabolism [van Buuren et al., 1972]. In mammals, the lethal dose of cyanide is in the range 0.5–3.5 mg/kg body weight [ATSDR, 1997]. Cyanide gas and certain salts are rapidly absorbed following inhalation and ingestion, but are more slowly absorbed by dermal exposure. Following inhalation, cyanide is rapidly distributed throughout the body, with measurable levels detected in blood and lungs, and death can occur within minutes. Reports of cyanide ingestion by humans and surveys of occupational exposure to cyanide indicate a plasma half-life of 20 min to 1 h after nonlethal exposures, during which cyanide is mainly converted into the 300-fold less toxic thiocyanate, a product primarily excreted in the urine (the oral LD50 of cyanide and thiocyanate in rats are 3 and 854 mg/kg, respectively) [ATSDR, 1997; Beasley and Glass, 1998]. Some microbes show high tolerance to a wide range of cyanide compounds, and have evolved multiple strategy to face with cyanide toxicity [Cooper et al., 2003; Fernández and Kunz, 2005; Raybuck, 1992]. Certain microbial taxa can colonize cyanide-polluted sites, having evolved degradative pathways for conversion of cyanide into less toxic metabolites. Exhaustive reviews addressing the biological degradation of cyanide compounds and the potential application of microorganisms in bioremediation of cyanide compounds have been published in recent years [Akcil, 2003; Baxter and Cummings, 2006; Ebbs, 2004]. Here, we combine current knowledge on microbial cyanide metabolism with novel insights into the role of bacterial rhodaneses in detoxification of this dangerous compound.

produced as a defensive metabolite [Vetter, 2000], as a virulence factor [Gallager and Manoil, 2001], or as part of the active iron-cyanide complexes of catalytic proteins such as NiFe-hydrogenases [Reissmann et al., 2003]. Plants are considered a main source of cyanide in the biosphere because they generate cyanide as co-product of ethylene (a hormone-like molecule) synthesis, or as a component of cyanogenic glycosides and cyanolipids. Over 2,500 plant species of families Fabaceae, Rosaceae, Linaceae, and Compositae (including agriculturally important species such as cassava, flax, sorghum, alfalfa, bamboo, peach, pear, cherry, plum, corn, potato, cotton, almond, and beans) are cyanogenic. Fruits and vegetables containing cyanogenic glycosides (amygdalin, linamarin, prunasin, dhurrin, lotaustralin, and taxiphyllin) can release cyanide upon enzymatic hydrolysis [Seigler, 1981; Vetter, 2000]. Cyanide production is a process commonly found also in fungi, while few prokaryotic species are known to be cyanogenic. Bacterial cyanogenesis appears to be restricted to Proteobacteria (Chromobacterium violaceum and fluorescent Pseudomonas species, including many strains of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas aureofaciens, and Pseudomonas chlororaphis) as well as to certain cyanobacteria [Krowels and Bunch, 1986]. In vivo studies have shown that cyanide production by bacteria is favored by addition of glycine to the growth medium [Lorck, 1948]. Glycine is the immediate metabolic precursor of cyanide, since HCN and CO2 are formed stoichiometrically by glycine oxidative decarboxylation [Castric, 1977; Laville et al., 1998; Wissing, 1974]. In experiments using radiolabeled [2-14C]glycine or [1-14C]glycine as the substrate, cyanide was found to be derived from the methylene carbon and CO2 from the carboxyl group of glycine in C. violaceum [Krowels and Bunch, 1986], P. fluorescens, and P. aeruginosa [Askeland and Morrison, 1983]. During this reaction, the C–N bond is retained, excluding possible alternative reactions like transamination or deamination [Pessi and Haas, 2004].

Anthropogenic Sources of Cyanide Natural Sources of Cyanide

Despite being a metabolic inhibitor, cyanide and chemically related compounds are synthesized, excreted and degraded in nature by hundreds of species of bacteria, algae, fungi, plants, and insects [Knowles, 1976]. It is 200

J Mol Microbiol Biotechnol 2008;15:199–211

Beyond natural sources, cyanide is massively produced by diverse industrial activities. Cyanide is produced on large scale worldwide (e.g. ⬃1 million tons in United States yearly; fig. 1) to fulfill the demand of major industrial countries. Two methods are commonly used for hydrogen cyanide production on industrial scale. The Cipollone /Ascenzi /Tomao /Imperi/Visca

Chemicals and allied products 19%

Primary metal industries 11%

Petroleum and coal products 5%

Metal mining 56%

Fabricated metal products 4% Electric, gas, and sanitary services 4%

Fig. 1. Industrial sectors with reported cy-

anide production-related waste in the entire United States. The contribution of each sector to total production is expressed as percentage. Data were retrieved from the web site http://www.scorecard.org/ chemical-profiles/rank-industrial.

Electronic and other electric equipment; Transportation equipment; Miscellaneous manufacturing industries; Instruments and related products; Stone, clay, and glass products; Industrial machinery and equipment
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