What if two hydrogenases are about eighty-five percent identical, yet one is far more likely to shut down in oxygen—and that apparently disadvantage helps it survive oxygen exposure? Hydrogenases are structurally diverse metalloenzymes that catalyze the reversible oxidation of dihydrogen.

[FeFe] hydrogenases contain a complex six-iron active cofactor called the H-cluster. These enzymes are of particular interest to industrial applications in hydrogen-producing biological and bio-hybrid systems. The figure’s right-hand diiron subcluster contains the vacant coordination position marked by the red arrow, next to the iron labeled with a cyanide ligand.

It also shows the nearby cysteine-derived sulfur connections and the bridging amine-containing ligand that link this subcluster to the larger six-iron framework. The genetic signatures that hallmark the oxygen tolerance of [FeFe] hydrogenases have yet to be resolved, creating a barrier to identifying similar, better-performing enzymes for bioengineered hydrogen-producing systems.

In CbHydA1, increased protein flexibility was proposed to allow Cys367 to adopt rotamers inaccessible to oxygen-sensitive enzymes, so the relationship between safe inactivation and amino acid sequence is expected to be complex. Site-specific amino acid substitutions affected oxygen tolerance in CbA5H, while a CbHydA1-PsaE fusion showed altered oxidative inactivation behavior in protein film voltammetry experiments.

Molecular dynamics simulations also suggested that hydrophobic residues near Cys367 are relevant, showing that oxygen tolerance cannot be attributed to a small subset of amino acid alterations. To understand the degree of sequence similarity required to retain oxygen tolerance, close homologues of CbHydA1 were investigated.

The study examines TgHydA1, a group A [FeFe] hydrogenase from Terrisporobacter glycolicus strain KPPR-9. Terrisporobacter glycolicus, previously known as Clostridium glycolicum, is an obligatory anaerobic, spore-forming, Gram-positive organism.

TgHydA1 shows a surprising divergence in the inactivation process despite about eighty-five percent sequence similarity with CbHydA1. Electrochemical assays show that the discrepancy originates phenomenologically from substantially altered reactivation energetics, which poises TgHydA1 toward the Hinact state under typical laboratory conditions.

TgHydA1 was cloned into the pET28a vector and transformed into E. coli BL21(DE3) delta iscR cells for heterologous protein production. The gene was augmented with affinity tags to facilitate purification, using a method previously used to produce CbHydA1. After isolation, the FeS clusters were reconstituted and the H-cluster was incorporated using a synthetic two-iron precursor.

Elemental analysis showed fifteen point four plus or minus one point two iron atoms per protein, suggesting successful reconstitution of the three FeS cofactors: two auxiliary four-iron, four-sulfur clusters and the H-cluster. A series of protein film voltammetry experiments was performed at various pH values to investigate this behavior.

In TgHydA1, hydrogen ion reduction current is disproportionately larger than hydrogen oxidation current even at high pH, where [FeFe] hydrogenases typically show the opposite behavior. The traces also show substantial hysteresis around the hydrogen ion and hydrogen thermodynamic equilibrium potential, and this hysteresis is more prominent at the higher scan rate of one hundred millivolts per second.

Compared with CbHydA1 under identical conditions, inhibition of the hydrogen oxidation current occurs at a less oxidizing potential, matching TgHydA1's higher tendency to inactivate in an oxidizing environment. Removing potassium chloride caused only marginal changes in the traces, confirming that chloride does not substantially affect the observed inactivation process.

In the central oxidation region, the red TgHydA1 trace reaches roughly forty microamps, while the blue CbHydA1 trace peaks near twenty microamps on the left current axis. The red curve’s forward and reverse branches also separate more strongly around the peak, whereas the blue branches form a narrower loop.

Chronoamperometry experiments repeatedly stepped CbHydA1 and TgHydA1 between more oxidizing, inactivating potentials and more reducing, activating potentials, beginning at a fully activating potential of minus eight hundred millivolts versus the normal hydrogen electrode. Analyzing the catalytic current after each potential step allows kinetic rates for interconversion between active and inactive states to be extracted.

The analysis used a model that assumes two active states in a dynamic equilibrium. The most notable result is that the reactivation rate values are universally about an order of magnitude lower for TgHydA1 than for CbHydA1 at any given potential.

This observation phenomenologically explains TgHydA1's propensity to remain in the Hinact state. The key result of the experiment is that, even without sodium dithionite, exposure of TgHydA1 to air was tested. Exposure to air results in only a minor loss of activity, less than twenty percent after three cycles, compared with the anaerobic control.

An increased propensity for inactivation in TgHydA1 therefore coincides with protein stability, at least regarding its interaction with oxygen. This trend warrants further investigation into why TgHydA1 shows greater stability upon aerobic inactivation and whether that translates into enhanced oxygen tolerance.

The protein's performance under aerobic conditions when coupled to a photosystem I complex remains a planned subject for future study. The ability to transition into the oxygen-stable state shows natural variation even between close [FeFe] hydrogenase homologues. This deviation is suggested to be controlled by the rate of enzyme reactivation, likely through the thermodynamic properties of the metallocofactors rather than structural mobility.

The propensity for inactivation is accompanied by increased stability upon oxygen exposure, encouraging closer examination of electron-transfer energetics within the protein. Because rapid reactivation is likely important for designing efficient hydrogen-producing systems, understanding the structure-function relationships that dictate oxygen tolerance is essential.

TgHydA1 shows that oxygen tolerance is not determined by sequence similarity alone: its much slower reactivation favors the inactive state, while exposure to air causes only a minor activity loss. That makes reactivation kinetics and metallocofactor energetics central design targets.