The oxochlorates are man-made biocides, bleaches, and oxidizers that, because of their extensive use, high solubility, and toxicity, are now serious contaminants of fresh water. Microbes were recently discovered that can detoxify these contaminants enzymatically, coupling their reduction to the generation of respiratory energy. The end product of the reduction pathway is chlorite (ClO2-), itself an EPA-regulated compound and widely used bleach and microbicide. Chlorite in these organisms is converted by the enzyme chlorite dismutase (Cld) to harmless Cl- and O2 via a heme-dependent, O-O bond forming mechanism which we have recently begun to elucidate. Heme enzymes catalyze an array of biologically essential reactions, where reaction specificity is dictated by the protein environment. The first objective of the proposed work is to define the structure-function relationships that allow a Cld from a model perchlorate respirer, Dechloromonas aromatica, to catalyze the decomposition of chlorite with extraordinary specificity. In forming our hypotheses, we have drawn on well documented structure-function models for heme peroxidases, which are expected to share active site but not catalytic or further structural similarities with Cld. Methods to be used in pursuing this objective include site directed mutagenesis, steady state and rapid kinetics, resonance Raman spectroscopy, and X-ray crystallography. The second objective is to elucidate the broader role of Clds in the hundreds of bacteria and even archaea where cld homologs have been found. It has been proposed that chlorite dismutation evolved relatively recently, in perchlorate-respiring bacteria, in response to the anthropogenic selection pressure applied by perchlorate pollution. The function of the ancestral cld gene product in non-perchlorate respirers is completely unknown, and likely unrelated to chlorite. Cld in these organisms is expected to play an important and potentially novel antioxidant role. In pursuit of this objective, Clds representing the two groups of cld sequences from non-respirers will be expressed. A series of chemical and kinetic experiments will be carried out, first to define the catalytic or sensor-regulator functions that each is capable of, and second to determine the efficiency with which the enzymes carry out these functions. The potential health impacts of this work are several: first, it provides essential knowledge and materials for bioremediation strategies against oxochlorates, or for biotechnological applications of Cld's O2 generating chemistry (e.g., for wound healing or water decontamination). Second, it supplies two kinds of fundamental information: about an entirely novel heme-catalyzed reaction, and about a widespread, highly conserved, and yet un-described microbial enzyme family that likely has an important antioxidant function. Finally, chlorite and related compounds are microbicides. This work consequently offers a paradigm for a new form of evolved anti-microbicide resistance.

Public Health Relevance

The oxochlorates are man-made, bleach-like chemicals that have recently become serious and widespread contaminants of fresh water. The proposed work will define critical features of the natural molecular systems used by certain microbes to detoxify oxochlorates, generating harmless Cl- and O2 gas. This work is essential for engineering efficient bioremediation strategies for the oxochlorates, as well as for other biotechnological applications of O2 generation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090260-03
Application #
8097222
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Anderson, Vernon
Project Start
2009-08-01
Project End
2012-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
3
Fiscal Year
2011
Total Cost
$244,140
Indirect Cost
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Geeraerts, Zachary; Rodgers, Kenton R; DuBois, Jennifer L et al. (2017) Active Sites of O2-Evolving Chlorite Dismutases Probed by Halides and Hydroxides and New Iron-Ligand Vibrational Correlations. Biochemistry 56:4509-4524
Celis, Arianna I; Gauss, George H; Streit, Bennett R et al. (2017) Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ). J Am Chem Soc 139:1900-1911
Streit, Bennett R; Celis, Arianna I; Shisler, Krista et al. (2017) Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b. Biochemistry 56:189-201
Streit, Bennett R; Kant, Ravi; Tokmina-Lukaszewska, Monika et al. (2016) Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway. J Biol Chem 291:862-71
Machovina, Melodie M; Usselman, Robert J; DuBois, Jennifer L (2016) Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations. J Biol Chem 291:17816-28
Celis, Arianna I; DuBois, Jennifer L (2015) Substrate, product, and cofactor: The extraordinarily flexible relationship between the CDE superfamily and heme. Arch Biochem Biophys 574:3-17
Celis, Arianna I; Geeraerts, Zachary; Ngmenterebo, David et al. (2015) A dimeric chlorite dismutase exhibits O2-generating activity and acts as a chlorite antioxidant in Klebsiella pneumoniae MGH 78578. Biochemistry 54:434-46
DuBois, Jennifer L; Ojha, Sunil (2015) Production of dioxygen in the dark: dismutases of oxyanions. Met Ions Life Sci 15:45-87
Sullivan, Sarah A; Streit, Bennett R; Ferguson, Ethan L et al. (2015) Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications. Anal Biochem 478:82-9
Celis, Arianna I; Streit, Bennett R; Moraski, Garrett C et al. (2015) Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ. Biochemistry 54:4022-32

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