Most hydrogen-making enzymes fail when they meet air. But two nearly matching versions of one enzyme behave very differently: one recovers quickly, while the other gets stuck in a protected inactive state. Most known hydrogen-making enzymes can operate only without oxygen, which significantly limits their industrial use.

A small group stays more stable in oxygen, renewing interest in them for renewable hydrogen production. The key protection is a rapid, reversible move into an oxygen-stable inactive state under oxidative conditions that would otherwise damage the enzyme.

That ability lets the enzyme operate on air while retaining some degree of oxygen tolerance, as demonstrated by aerobic hydrogen production. The everyday picture is a machine with a safe parking mode: it stops working temporarily when danger arrives, then can switch back instead of being permanently broken.

The genetic signs of oxygen tolerance remain unresolved, creating a barrier to finding similar enzymes for improved biological hydrogen systems. The relationship between safe inactivation and amino acid sequence is expected to be complex.

Changes on the protein surface affected oxygen tolerance, and altered behavior also emphasized the importance of the protein’s overall movements. Earlier work therefore suggests that oxygen tolerance cannot be attributed to a small subset of amino acid alterations.

To understand how much sequence similarity is needed to retain oxygen tolerance, the study examined a close relative of the better-characterized enzyme. That close relative is a group A iron-iron hydrogenase from Terrisporobacter glycolicus, a spore-forming, Gram-positive organism that normally lives without oxygen.

Despite high sequence similarity with the comparison enzyme, the Terrisporobacter enzyme showed a surprising difference in how it became inactive. The difference came from a substantially altered return-to-activity process, which favored the oxygen-stable inactive state under typical laboratory conditions.

At high pH, one hydrogen-producing enzyme still spends much of its activity making hydrogen rather than breaking it down, unlike the usual balance for this enzyme family. That unusual imbalance helps explain why it later remains trapped in an inactive state.

The researchers switched the two enzymes between conditions that encourage inactivation and conditions that encourage activation. The resulting current changes revealed rates for moving between active and inactive states. The analysis treated the enzymes as having two active states that can continually exchange with one another.

The most notable result is that the return-to-activity rate was about ten times lower for the Terrisporobacter enzyme than for the comparison enzyme at every tested condition. Because its return-to-activity rate was about an order of magnitude lower at every tested potential, the Terrisporobacter enzyme tended to remain in the inactive Hinact state.

The key result of this experiment is that exposing the Terrisporobacter enzyme to air produced the observed result even without the added chemical used to support the test. The ability to enter an oxygen-stable state varies naturally even between close relatives.

The study suggests that the difference is controlled by how quickly the enzyme becomes active again, likely through the behavior of its metal-containing parts rather than its structural movement. The tendency to become inactive is accompanied by greater stability during oxygen exposure, while rapid reactivation is likely important for designing efficient hydrogen-producing systems.

For anyone hoping to use biology to make renewable hydrogen, finding an enzyme that survives air is not enough; its reactivation rate and stability also matter. The study therefore points toward examining electron-transfer energetics and structure-function relationships when developing efficient hydrogen-producing systems with oxygen tolerance.

The difference is not simply the enzyme’s overall shape or sequence. A slower return to activity makes one version more air-stable, a clue that could help build renewable hydrogen systems that keep working outside oxygen-free laboratories.