Kinetic Modeling of the Reversible or Irreversible Electrochemical Responses of FeFe-Hydrogenases

Abstract

The enzyme FeFe-hydrogenase catalyzes H 2 evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe 2 (CO) 3 (CN) 2 (azadithiolate)] subsite. Previous investigations of its mechanism were mostly conducted on a few "prototypical" FeFehydrogenases, such as that from Chlamydomonas reinhardtii(Cr HydA1), but atypical hydrogenases have recently been characterized in an effort to explore the diversity of this class of enzymes. We aim at understanding why prototypical hydrogenases are active in either direction of the reaction in response to a small deviation from equilibrium, whereas the homologous enzyme from Thermoanaerobacter mathranii (Tam HydS) shows activity only under conditions of very high driving force, a behavior that was referred to as "irreversible catalysis". We follow up on previous spectroscopic studies and recent developments in the kinetic modeling of bidirectional reactions to investigate and compare the catalytic cycles of Cr HydA1 and Tam HydS under conditions of direct electron transfer with an electrode. We compare the hypothetical catalytic cycles described in the literature, and we show that the observed changes in catalytic activity as a function of potential, pH, and H 2 concentration can be explained with the assumption that the same catalytic mechanism applies. This helps us identify which variations in properties of the catalytic intermediates give rise to the distinct "reversible" or "irreversible" catalytic behaviors.

Outer-sphere effects on the O2 sensitivity, catalytic bias and catalytic reversibility of hydrogenases

“…The separation between the two catalytic waves (the difference, for a given enzyme, between the values of the two catalytic potentials, the midpoint potentials of the H 2 oxidation and H + reduction catalytic waves) is related to the difference between the redox potentials of the two transitions of the active site, although the catalytic potentials are shifted from the thermodynamic values, just like Michaelis constants depart from the true dissociation constants. 22,32,110 This implies that the more stable the half reduced state of the active site (the so-called Hred state), the more irreversible the response; this appears to be the main reason why the enzyme from Tam behaves very irreversibly, 33 although it is still unknown which residues in the environment of the active site make the half reduced state very stable. 111 That the proton relay near the H-cluster has a lower p K a in Tam than in Cr also contributes to making the response of the former less reversible.…”

“… 111 That the proton relay near the H-cluster has a lower p K a in Tam than in Cr also contributes to making the response of the former less reversible. 33 …”

“…The turnover frequency depends on the electrode potential (and other experimental parameters such as T, pH, and [H 2 ]) and this dependence informs on the properties of the intermediates in the catalytic cycle. 32,33 The second term in the right-hand side of eqn (1), the surface concentration of the active enzyme, is smaller than the total amount of enzyme adsorbed on the electrode, first because some enzyme molecules may be adsorbed in a configuration that does not allow electrical communication with the electrode (and hence do not contribute to the current), and second because some enzyme molecules may (in)activate in a certain range of electrode potentials. If such a redox-driven (in)activation process is slow on the time scale of the experiment, it results in a hysteresis in the voltammetric signature.…”

Catalytic Bias and Redox-Driven Inactivation of the Group B FeFe Hydrogenase CpIII

“…For example, the group A “CbA5H″ FeFe hydrogenase from Clostridium beijerinckii (Cb) is protected from oxygen damage by the binding to the distal Fe ion of the conserved cysteine that is the first proton relay; this binding can occur because nonconserved residues that are remote from the active site make the protein loop that bears the cysteine residue more flexible than in other hydrogenases. ,, In the group D FeFe hydrogenase from Thermoanaerobacter mathranii, protons are transferred along a pathway that is entirely distinct from that of prototypical hydrogenases, , and catalysis is ‘irreversible’, that is, H 2 oxidation and production only occur at the price of a large thermodynamic driving force. , …”

Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility

“…Hydrogenases are excellent exemplars of reversible electrocatalysis, their inherent activities being comparable with Pt metals. , Moreover, they have long been important subjects for protein film electrochemistry (PFE), a suite of techniques providing exquisite, complementary information on redox enzymes. − For [FeFe]-hydrogenases, much is now established about the mechanism of H 2 activation by the active-site H-cluster. , Experimentally, the specific role of concerted PCET in electrocatalytic reversibility is difficult to isolate among other contributing factors: , however, for two [FeFe]-hydrogenases, mild disruption of a remote proton-transfer pathway by exchanging a Glu for Asp caused the reversible electrocatalytic trace to become sigmoidal (an inflection appearing in the otherwise continuous potential dependence) . Adapting the concept shown in Figure , the observations, interpreted as retarded proton mobility, confirmed that long-range concerted PCET underpins the reversibility of [FeFe]-hydrogenases.…”