Lyophilization protects [FeFe]-hydrogenases against O(2)-induced H-cluster degradation

Nature has developed an impressive repertoire of metal-based enzymes that perform complex chemical reactions under moderate conditions. Catalysts that produce molecular hydrogen (H(2)) are particularly promising for renewable energy applications. Unfortunately, natural and chemical H(2)-catalysts are often irreversibly degraded by molecular oxygen (O(2)). Here we present a straightforward procedure based on freeze-drying (lyophilization), that turns [FeFe]-hydrogenases, which are excellent H(2)-producers, but typically extremely O(2)-sensitive in solution, into enzymes that are fully resistant against O(2). Complete dryness protects and conserves both, the [FeFe]-hydrogenase proteins and their inorganic active-site cofactor (H-cluster), when exposed to 100% O(2) for days. The full H(2)-formation capacity is restored after solvation of the lyophilized enzymes. However, even minimal moisturizing re-establishes O(2)-sensitivity. The dry [FeFe]-hydrogenase material is superior also for advanced spectroscopic investigations on the H-cluster reaction mechanism. Our method provides a convenient way for long-term storage and impacts on potential biotechnological hydrogen production applications of hydrogenase enzymes.