Carnegie Mellon University
September 21, 2023

Researchers Find Unusual Feature in Enzyme

By Heidi Opdyke

Jocelyn Duffy
  • Associate Dean for Communications, MCS
  • 412-268-9982

Researchers at Carnegie Mellon University and collaborators have identified a short-lived reactive intermediate that is used to perform a key step in the process to make the antioxidant ovothiol A (OSHA). The work could have applications for potential future treatments of aging-related diseases and liver cancer.

OSHA is derived from free amino acids, histidine and cysteine, via an ovothiol biosynthetic pathway, where OvoA catalyzes the key step in OSHA biosynthesis. OvoA is a nonheme iron enzyme found in sea urchins, other marine invertebrates and the bacteria Methyloversatillis thermotolerant.

"OSHA acts as a type of free radical scavenger. These compounds protect the organism from damaging free radicals produced during oxidative stress," said Jared Paris, a graduate student in chemistry who is first author on a paper about the work in Angewandte Chemie. "Specifically, within sea urchins, OSHA is produced during egg fertilization to make sure the creature doesn't have any free radicals generated during the energy intensive fertilization process.

Nonheme iron enzymes are a family of proteins that use iron but lack the heme molecule found in most living cells. Heme is an organic compound that contains an iron atom that when combined with globin proteins forms hemoglobin, which carries oxygen in red blood cells around the body. Nonheme iron enzymes catalyze a broad range of chemical transformations, and Paris said that products from some of those reactions can have health care implications.

"Fundamental metalloenzymes (proteins with metal ions) have such a wide variety of chemistries. Yet with all their types of chemistry — especially the nonheme ones — very little is known about what drives these reactions," Paris said. "There's rich, rich chemistry associated with those that could lead to drug designs or natural products."

For example, OSHA inhibits liver cancer cell proliferation and could have potential to be used in cancer treatments. A similar small molecule, ergothioneine (ESH), shows protective roles in diseases such as dementia, depression, atherosclerosis, cardiovascular disorders and nonalcoholic fatty liver disease.

Paris spent several years identifying and characterizing "intermediate 490," the first experimentally observed intermediate spin iron(IV) species in OvoA. Iron(IV) refers to iron in a high oxidation state where iron bonds to amino acids in a protein structure. Through the process, electrons are removed from the iron molecule. As the iron tries to recapture its electrons, it gains more reactivity.

Spin states of molecules refers to how the iron atom's electrons are configured, and spin can be "high" (S = 2), "intermediate" (S = 1) or "low" (S= 0) for iron(IV) species.

In this case, the process to form OSHA used an S = 1 iron(IV) intermediate as the mechanism that enabled carbon-sulfur bond formation in the enzyme OvoA. The accepted knowledge was that in nature, there are no enzymatic systems with an S = 1 configuration.

"In native enzymes, all of the iron(IV) species being discovered were S = 2," said Yisong (Alex) Guo, associate professor of chemistry who advises Paris and leads the Guo Group. "This thinking needs to be modified. It will be interesting to further explore the reactivities of this newly identified S = 1 iron(IV) species found in OvoA."

The enzyme was provided by Boston University, and experiments to support the work were run at the Stanford Synchrotron Radiation Lightsource at the SLAC National Accelerator Laboratory and the Penn State Radiation Science and Engineering Center.

The work is supported by the National Science Foundation, the National Institutes of Health.

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