Ever since it flooded our planet’s atmosphere more than 2.1 billion years ago, oxygen has played a starring role in our lives. But despite its crucial importance to the function of all living organisms, researchers have not been able to study the details of how mitochondria and other parts of living cells use it. They can crank up or dial down oxygen levels in the cells’ environment, but there is currently no way to selectively change oxygen levels inside individual cells, explains Vamsi K. Mootha, a molecular biologist at Harvard Medical School.
A new genetically encoded system developed in Mootha’s lab allows researchers to provide puffs of oxygen to specific parts of a cell on demand (Proc. Natl. Acad. Sci. USA 2022, DOI: 10.1073/pnas.2207955119). The tool will help researchers study precisely how oxygen levels affect metabolic processes—and according to the researchers, its future iterations might one day enable gene therapy that can deliver oxygen to damaged cells.
The first step in developing the system was to identifya protein that could be genetically encoded and that spurs oxygen production inside the cell. At first, the researchers struggled to think of a naturally occurring system that produces molecular oxygen (O2) and was simple enough to deploy to a human cell. But then Mootha read about enzymes present in many types of bacteria and archaea called chlorite O2-lyases, or chlorite dismutase (Cld) enzymes, which convert chlorite (ClO2-) into molecular oxygen and chloride ions. In these anaerobic organisms, chlorite is a metabolic by-product that Cld enzymes clear away.
Mootha’s team decided to use them as a chassis for their oxygen-producing system. The researchers screened multiple Cld enzymes to identify those that could be expressed at high levels in human cells, and then, after some chemical fiddling, ended up with a usable sequence. They tested the protein to make sure it could function inside cells.
Then, because the chlorite needed to set off the reaction does not readily enter cells, the researchers screened for transporters that could carry it in. They homed in on a human sodium iodide transporter, normally expressed in the thyroid, that could do the job. When the cell expresses both the Cld enzyme and the sodium iodide transporter, the reaction switches on in the presence of chlorite.
They also added a targeting sequence to the Cld enzyme, sending it either to a cell’s mitochondria or to the cytosol. “It’s an important proof of concept that we are able to localize oxygen generation in human cells, and we are pretty excited about that,” Mootha says.
The team “did all of the right controls to show they are making O2 in the way they say they are going to do,” says Amy Palmer, a biochemist at the University of Colorado Boulder, who calls the work “a really clever idea.” The only limitation, she says, is the tool’s complexity, because it involves genetically encoding two different components.
So far, the construct produces puffs of oxygen amounting to about 10% of the oxygen that cells normally generate, and the puffs last on the order of minutes to about half an hour, Mootha says. But ultimately, the group hopes to engineer a longer-acting system or to identify transporters with higher activity.