Investigation of the Unusual Ability of the [FeFe] Hydrogenase from Clostridium beijerinckii to Access an O₂-Protected State

Abstract

[FeFe] hydrogenases are enzymes capable of producing and oxidizing H2 at staggering submillisecond time scales. A major limitation in applying these enzymes for industrial hydrogen production is their irreversible inactivation by oxygen. Recently, an [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) was reported to regain its catalytic activity after exposure to oxygen. In this report, we have determined that artificially matured CbHydA1 is indeed oxygen tolerant in the absence of reducing agents and sulfides by means of reaching an O2-protected state (Hinact). We were also able to generate the Hinact state anaerobically via both chemical and electrochemical oxidation. We use a combination of spectroscopy, electrochemistry, and density functional theory (DFT) to uncover intrinsic properties of the active center of CbHydA1, leading to its unprecedented oxygen tolerance. We have observed that reversible, low-potential oxidation of the active center leads to the protection against O2-induced degradation. The transition between the active oxidized state (Hox) and the Hinact state appears to proceed without any detectable intermediates. We found that the Hinact state is stable for more than 40 h in air, highlighting the remarkable resilience of CbHydA1 to oxygen. Using a combination of DFT and FTIR, we also provide a hypothesis for the chemical identity of the Hinact state. These results demonstrate that CbHydA1 has remarkable stability in the presence of oxygen, which will drive future efforts to engineer more robust catalysts for biofuel production.

A safety cap protects hydrogenase from oxygen attack

“…3 and 5). This observation is consistent with a recently published hypothesis according to which the loop that bears C367 in CbA5H may be flexible enough to allow the binding of the cysteine to Fe d 31 . The strictly conserved cysteine residue is involved in long-range proton transfer to and from the H-cluster (Supplementary Figure 6) 4,16,18 .…”

“…However, DdH becomes O 2 -sensitive after gaining catalytic activity 10,19 . For DdH and some other [FeFe]-hydrogenases, sulfide-addition under oxidizing conditions (either aerobic or anaerobic) also produces the H inact -state; the anaerobic formation of H inact is detected in cyclic voltammetry by the early onset of oxidative inactivation 30,31 . Rodríguez-Maciá et al concluded that H inactformation under oxidizing conditions is the consequence of sulfide-binding to Fe d 30 , but this protected state is also formed in CbA5H 27,31 and CpIII ([FeFe]-hydrogenase III from Clostridium pasteurianum) 32 in the absence of exogenous sulfide.…”

“…For DdH and some other [FeFe]-hydrogenases, sulfide-addition under oxidizing conditions (either aerobic or anaerobic) also produces the H inact -state; the anaerobic formation of H inact is detected in cyclic voltammetry by the early onset of oxidative inactivation 30,31 . Rodríguez-Maciá et al concluded that H inactformation under oxidizing conditions is the consequence of sulfide-binding to Fe d 30 , but this protected state is also formed in CbA5H 27,31 and CpIII ([FeFe]-hydrogenase III from Clostridium pasteurianum) 32 in the absence of exogenous sulfide. The sulfideindependent, reversible transition between H ox and the inactive, O 2 -resistant H inact -state is therefore an uncharacterized and intriguing feature of certain [FeFe]-hydrogenases, which significantly enhances their utilization potential (Fig.…”

Successes, challenges, and opportunities for quantum chemistry in understanding metalloenzymes for solar fuels research

“…29 Following the development of a computational methodology for an accurate prediction of the IR spectra of the active site of [FeFe]-hydrogenase, 256 many studies have been conducted using DFT calculations and helped in the unambiguous assignment of the structure and redox states of intermediates like for the H-cluster. [257][258][259][260] While the calculated absolute vibrational frequencies are often not quantitatively accurate, shifts in frequencies due to protonation and/or reduction events are reliable. Due to the peculiar vibrational frequencies of the CN and CO groups, it is possible to distinguish the redox states of the [NiFe] active site.…”

A Beginner’s Guide to Thermodynamic Modelling of [FeFe] Hydrogenase

“…For a long time, the major drawback of [FeFe] hydrogenases was their oxygen sensitivity [8][9][10]. However, methods that have recently been identified can protect these enzymes for later use [11][12][13][14], and even enzymes with a self-protection mechanism have been discovered [15,16].…”