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ToxSci Advance Access published online on April 1, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp066
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© The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

CLONING, EXPRESSION AND BIOCHEMICAL PROPERTIES OF CPOX4, A GENETIC VARIANT OF COPROPORPHYRINOGEN OXIDASE THAT AFFECTS SUSCEPTIBILITLY TO MERCURY TOXICITY IN HUMANS

Tingting Li and James S. Woods

Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA

Author whom comments and page proofs should be sent: James S. Woods, Ph.D., Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98105, Telephone: 206 685-3443; Fax 206 685-4696, e-mail: jwoods{at}u.washington.edu

Received January 29, 2009; revision received March 9, 2009; accepted March 23, 2009


  Abstract

Coproporphyrinogen oxidase (CPOX) catalyzes the 2-step decarboxylation of coproporphyringen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg2+ present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was two-fold lower than that of CPOX (CPOX Km = 0.30µM, Vmax = 0.52 pmol protoporphyrin-IX; CPOX4 Km = 0.54µM, Vmax = 0.33 pmol protoporphyrin-IX). Hg2+ specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40-50%) than those of either wildtype or heterozygous. Additionally, enzymes from mutant, heterozygous and wildtype livers were comparably inhibited by Hg2+ (10 µM), decreasing CPOX4 activity to 25% that of the wildtype enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.

Key Words: Coproporphyrinogen oxidase; polymorphism; CPOX4; Hg.

Tingting Li, e-mail: litt{at}u.washington.edu; telephone: 206 685-1938


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