Redox regulation in O2 -tolerant [FeFe] hydrogenases: insights from two homologues.

Abstract

O₂-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H₂-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H₂-producing activity under oxygenic conditions. However, the genetic signatures of the O₂-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O₂-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1)-a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O₂-stable H_inact state, a hallmark of O₂ tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the H_inact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O₂-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O₂ that encompasses the protein environment and properties of the auxiliary metallocofactors.