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Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

ABSTRACT: Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO(2) (-)) to Cl(-) and O(2). The ability of Cld to promote O(2) formation from ClO(2) (-) is unusual. Heme enzymes generally utilize ClO(2) (-) as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO(2) (-)) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO(2) (-) within the distal pocket generates hypochlorite (ClO(-)) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO(-) with compound I forming the Cl(-) and O(2) products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O(2) may have a preferential direction for exiting the active site.

SUBMITTER: Goblirsch BR

PROVIDER: S-EPMC2909366 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

Goblirsch Brandon R BR Streit Bennett R BR Dubois Jennifer L JL Wilmot Carrie M CM

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20100413 6

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO(2) (-)) to Cl(-) and O(2). The ability of Cld to promote O(2) formation from ClO(2) (-) is unusual. Heme enzymes generally utilize ClO(2) (-) as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO(2) (-)) was determined to investigate f ...[more]

PMID: 20386942

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Project description:The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2 -) to yield Cl- and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2 -) with the evolution of O2 (k cat = (2.0±0.2)×102 s-1; k cat/K M = (1.2±0.2)×105 M-1 s-1 at pH 5.2). Stopped-flow studies of this BrO2 - decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK a of 6.7±0.1 in good agreement with the pK a of DaCld activity with ClO2 -. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pK a 6.7. Examination of reaction mixtures comprising DaCld and ClO2 - by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO2, which forms in preference to the analogous to AA• in the BrO2 - reaction. Addition of ClO- to Cpd II did not yield O2. Together these results are consistent with heterolytic cleavage of the O-BrO- and O-ClO- bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

Project description:Salinity and pH have direct and indirect impacts on the growth and metabolic activities of microorganisms. In this study, the effects of salt and alkaline stresses on the kinetic balance between nitrous oxide (N2O) production and consumption in the denitrification pathway of Dechloromonas aromatica strain RCB were examined. N2O accumulated transiently only in insignificant amounts at low salinity (≤0.5% NaCl) and circumneutral pH (7.0 and 7.5). As compared to these control conditions, incubation at 0.7% salinity resulted in substantially longer lag phase and slower growth rate, along with the increase in the amounts of transiently accumulated N2O (15.8 ± 2.8 μmoles N2O-N/vessel). Incubation at pH 8.0 severely inhibited growth and resulted in permanent accumulation of 29.9 ± 1.3 μmoles N2O-N/vessel from reduction of 151 ± 20 μmoles NO3 -/vessel. Monitoring of temporal changes in nirS 1, nirS 2, and nosZ transcription suggested that the nosZ/(nirS 1+nirS 2) ratios were indicative of whether N2O was produced or consumed at the time points where measurements were taken. The salt and alkaline stresses altered the N2O consumption kinetics of the resting D. aromatica cells with expressed nitrous oxide reductases. The N2O consumption rates of the cells subjected to the salt and alkaline stress conditions were significantly reduced from 0.84 ± 0.007 μmoles min-1 mg protein-1 of the control to 0.27 ± 0.02 μmoles min-1 mg protein-1 and 0.31 ± 0.03 μmoles min-1 mg protein-1, respectively, when the initial dissolved N2O concentration was 0.1 mM. As the rates of N2O production from NO2 - reduction was not significantly affected by the stresses (0.45-0.55 μmoles min-1 mg protein-1), the N2O consumption rate was lower than the N2O production rate at the stress conditions, but not at the control condition. These results clearly indicate that the altered kinetics of expressed nitrous oxide reductase and the resultant disruption of kinetic balance between N2O production and consumption was another cause of enhanced N2O emission observed under the salt and alkaline stress conditions. These findings suggest that canonical denitrifiers may become a significant N2O source when faced with abrupt environmental changes.

Dataset Information

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

Publications

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

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