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Cloning, Expression, and Characterization of Chlorite Dismutase from Dechloromonas aromatica RCB

EPA Grant Number: F07D10663
Title: Cloning, Expression, and Characterization of Chlorite Dismutase from Dechloromonas aromatica RCB
Investigators: Streit, Bennett
Institution: University of Notre Dame
EPA Project Officer: Manty, Dale
Project Period: September 1, 2007 through September 1, 2010
RFA: STAR Graduate Fellowships (2007)
Research Category: Hazardous Waste/Remediation , Fellowship - Chemistry and Biochemistry , Academic Fellowships

Description:

Objective:

Chlorite (ClO2-) is a widely used, highly soluble, and toxic bleaching agent. With its large scale use chlorite contamination of drinking water supplies has become a topic of concern. High doses of chlorite are believed to cause anemia as well as inhibit neurological development of young children. Therefore, means for detoxifying environmental chlorite are desirable. Nature however has already evolved an enzyme, chlorite dismutase (Cld), which catalyzes the dismutation of the toxic ClO2- into the benign products of chloride (Cl-) and molecular oxygen (O2). This reaction is interesting from a biological and biochemical point of view. Heme enzymes catalyze a large variety of enzymatic reactions, but none catalyze an O-O bond forming reaction. Also, while the redox potential of chlorite is favorable, the enzyme is used to specifically dismutate ClO2- with no net contribution to the formation of an electrochemical gradient. In this respect it is unlike perchlorate reductase (Pcr), the enzyme upstream in the chlorooxide respiration pathway. By studying the structural and electronic properties as well as the kinetics of Cld, an understanding of the mechanism of chlorite dismutation and the features distinguishing this from all other heme enzymes can be determined. A good understanding of how this enzyme functions will allow for its future use in sensing and remediation of chlorite contaminated water sources.

Approach:

In order to gain an understanding of the means by which Cld catalyzes chlorite dismutation, kinetic, spectroscopic, and structural techniques are being utilized. Through heterologous expression of the gene encoding Cld from D. aromatica, large amounts of soluble, active, fully heme incorporating enzyme are obtainable. The kinetics of chlorite dismutation can be monitored both through chlorite loss (by a developed iodometrically based discontinuous assay) as well as for O2 evolution (using a platinum oxygen-sensing electrode) to determine the steady state kinetic parameters KM, kcat, and kcat/KM. How factors such as ionic strength, pH, viscosity, inhibitors, and mutations of active site residues affect these catalytic parameters can give important information on the relationships between the active site structure and the reactivity of the enzyme. Through the use of kinetic techniques as well as spectroscopy and X-ray crystallography, Cld can be compared to other heme b enzymes providing a better understanding of how the physical and electronic active site structure affect the reactivity of Cld.

Expected Results:

The long-term goal of this project is to obtain a thorough understanding of the chemical, physical, and structural properties of chlorite dismutase. To this end, the steady-state kinetics of chlorite dismutase, including the roles of pH and product inhibition are being studied. In addition, spectroscopic and X-ray crystallographic methods are being applied to understand its geometric and electronic structure. Finally, factors relating to the enzyme’s stability in the presence of its strongly oxidizing substrate are being studied, in order to understand how its lifetime may be extended through enzyme modification. This work directly supports the development of enzymatic or whole-cell approaches to remediation and sensing of chlorite contaminated water sources.

Project Synopsis:

The heme enzyme chlorite dismutase (Cld) catalyzes the dismutation of chlorite, a groundwater contaminant, into Cl- and O2. What distinguishes Cld form other heme enzymes as well as how the enzyme carries out this difficult and novel O-O bond forming reaction is unknown. Using steady state kinetics as well as spectroscopic and structural characterization techniques we hope to gain an understanding of the mechanisms of chlorite dismutation and enzymatic inactivation during catalytic turnover of chlorite in hopes that the enzyme can be applied in remediation and sensing of chlorite contaminated water sources in the future.

Supplemental Keywords:

chlorite, heme, chlorooxide, peroxidase, catalase, heterologous expression, drinking water contamination, water remediation, sensing, steady state kinetics, spectroscopy, crystallography, oxygen, anemia, neurological, , Scientific Discipline

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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