Oxygen Tolerance in the [FeFe] Hydrogenase from Clostridium beijerinckii CbHydA1

Restricted (Penn State Only)
- Author:
- Corrigan, Patrick
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 06, 2022
- Committee Members:
- Philip Bevilacqua, Program Head/Chair
Carsten Krebs, Major Field Member
Squire Booker, Major Field Member
Alexey Silakov, Chair & Dissertation Advisor
John Golbeck, Outside Unit & Field Member - Keywords:
- Bioinorganic chemistry
hydrogenase - Abstract:
- [FeFe] hydrogenases are (bidirectional) enzymes capable of producing and oxidizing H2 at staggering sub-millisecond rates. A major limitation in applying these enzymes for industrial hydrogen production is their sensitivity to 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 apo-CbHydA1 activated with a synthetic [2Fe]H subcluster 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 hours 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. Furthermore, we go on to characterize the electronic structure of the Hox and Hox-CO states using EPR methods. The purpose of this study is to determine if there are differences in the electronic structure of CbHydA1 as compared to other known [FeFe] hydrogenases that enable this unique enzyme to enter the Hinact state. We show that both the Hox and Hox-CO state are composed of two species. Through FTIR and continuous wave EPR, we show that these species do not arise from the occurrence of an HoxH+ like-state but are nevertheless pH-dependent. Using HYSCORE spectroscopy and site-specific isotope labeling, we demonstrate that these species have the same hyperfine coupling values across the [2Fe]H subcluster. We also show that the electron density across the [2Fe]H subcluster is not shared equally. Finally, our findings suggest that structural changes give rise to the different species observed and cause dramatically different EPR signals for each state. In our final investigation we search for possible intermediates in the conversion of the Hox to Hinact state via photodissociation at cryogenic temperatures. Additionally, we show that the Hox-CO state converts to the Hox state as well as the light-induced state, HLI. Photodissociation of the Hinact state results in a newly characterized state termed the oxidized light state, or HOL. The FTIR stretching frequencies of this new state suggest that the enzyme is reduced by one electron between these state. We propose multiple possibilities for the source of this extra reducing equivalent. All of these results taken together demonstrate that CbHydA1 has remarkable stability in the presence of oxygen, the unique features of its catalysis, and a possible mechanism of inactivation. These results will drive future efforts to engineer more robust catalysts for biofuel production.