2024 FrenchBIC thesis prize to Andrea Fasano

Two thesis prizes were awarded in 2024, to Andrea Fasano and Afridi Zamader.

Andrea Fasano

During my Ph.D. at the Bioénergétique et Ingénierie des Protéines laboratory (BIP), in Christophe Léger’s team, I studied metallo-enzymes, called hydrogenases, that catalyse the redox reaction of hydrogen oxidation and production. Despite the great biodiversity of hydrogenases in Nature, the focus has mainly been on just a few of them, called “standard”. Only recently have new, “atypical” hydrogenases started to be characterised.

My thesis aimed to deepen our knowledge on the hydrogenase’s biodiversity. I have investigated different  “atypical” hydrogenases, using Protein Film Electrochemistry to characterise their catalytic properties, such as directionality (the ability to catalyse the reaction in only one or both directions); reversibility (related to how much thermodynamic driving force is required to catalyse the reaction in either direction); and tolerance to inhibitors, such as O2. An example of the catalytic diversity of [FeFe]-hydrogenases in voltammetry is shown in figure 1.

Two main families of hydrogenases are defined by the metal content of their active sites: [FeFe] and [NiFe]. Enzymes from the same family have the same active site, but may have very different catalytic properties. Comparing different enzymes allowed us to elucidate the role of the protein scaffold in determining the different catalytic properties.

Regarding [FeFe]-Hydrogenases, I have studied three atypical enzymes. In a first project, I have characterised the hydrogenase of Thermoanaerobacter marthranii (Tam HydS), as it is the only hydrogenase giving an irreversible catalytic response (figure 1C). In our first work, we concluded that it is an intrinsic property of this enzyme (it is not a result of electron transfer with the electrode being slow), and we proved that its catalytic cycle is of the same kind as that of standard, reversible [FeFe]-hydrogenases (figure 1A and 1B) (Fasano et al, JACS 2021). Reversibility is a very important property for any catalyst, as it prevents the dissipation of thermodynamic driving energy during catalysis. To elucidate the molecular determinants behind reversible or irreversible responses, we developed a kinetic model that relates the catalytic response to the details of the steps of the catalytic cycle of the enzyme. We fitted this model to the catalytic responses of Tam HydS and of a standard, reversible, hydrogenase. This comparison allowed us to understand which steps of the catalytic cycle determine the reversibility of the catalytic response (Fasano et al, Bioelectrochemistry 2023, Fasano et al, JACS 2024). The following challenge is now to understand which aminoacids affects those steps and how, in order to really tune the reversibility of the enzyme.

Figure 1: The electrochemical diversity of FeFe hydrogenases, illustrated by catalytic voltammograms recorded under conditions of direct electron transfer and using a rotating disc electrode, all under one atm. of H2. A: C. reinardtii HydA1. B: C. acetobutylicum hydrogenase I. C: Thermoanaerobacter mathrani HydS. D: C. pasteurianum hydrogenase II. E: C. beijerinckii hydrogenase (CbA5H), and the C367D variant. F: variants of CbA5H, A561F (green); P386L (blue); L364F (dark red); M382E (orange). G: C. pasteurianum hydrogenase III and M. elsdenii hydrogenase II. H: a variant of C. pasteurianum hydrogenase III (Fasano et al, Chem. Sci 2024).

In a second project I could also work on the only O2 tolerant [FeFe]-hydrogenase known so far, that from Clostridium beijerinckii (CbA5H) (orange trace in figure 1E). We elucidated the kinetic reasons behind the increased tolerance toward O2 induced by the replacement of a residue 18 Å from the active site (Rutz, Das, Fasano et al, ACS catalysis 2022). Intrigued by the similar catalytic response between CbA5H and the third hydrogenase from Clostridium pasteurianum (CpIII) (darkgreen in figure 1G), belonging to a very poorly studied group of [FeFe]-hydrogenases, we have investigated the oxidative inactivation of CpIII and discussed it in terms of O2 protection. Moreover, our results suggest that CpIII and other related hydrogenases are part of a specific subgroup, defined by a particular protein environment close to the active site (Fasano et al, BioRxiv, ACS Cat. 2024 (in press)).

Regarding the family of the [NiFe]-hydrogenases, I have worked on two enzymes that have very similar aminoacidic sequences, yet very different properties in terms of O2-tolerance and directionality. These enzymes are made of two subunits, one contains the active site and the other a chain of FeS clusters for electron transfer. Mutagenesis of single aminoacids proved inconclusive to understand the molecular determinants of these catalytic properties. Therefore, we aimed to first understand the role of each of the two subunits. To achieve this goal we used a completely new strategy: we engineered a catalytically active chimeric complex made of one subunit from each enzyme. Comparing the responses of this chimeric hydrogenase with the WT enzymes allowed us to elucidate which subunit is responsible for each catalytic property (Fasano et al, JACS 2023). The chimeric design of hydrogenases opens many doors to engineer enzymes combining desired properties, such as O2 tolerance and the bias towards a specific direction of the reaction.

Current affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, Aix Marseille Université, Marseille, France.
Past affiliations:
Master thesis: D. Carbonera and P. Constantini teams, Department of Chemical Science and Department of Biology, respectively; University of Padova.
Bachelor thesis: A. Danielli team, Department of Pharmacy and Biotechnology, University of Bologna.