Proton-Coupled Electron Transfer Dynamics in the Catalytic Mechanism of a [NiFe]-Hydrogenase

Abstract

The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase

“…Previously, we have shown that the reverse EPT process, probed by photoreduction of Ni a -S, correlates with pH in the same way, in both rate and KIE. 33 These observations suggest a common residue responsible for the forward and reverse EPT, with both mechanisms involving significant nuclear tunneling. 33 It is clear from the kinetic data that at least one Ni a -I state is an intermediate in the conversion of Ni a -S and Ni a -C. Three potential mechanisms could explain the connection of the Ni a -I states to Ni a -S and Ni a -C: (1) a serial mechanism in which both Ni a -I 1 and Ni a -I 2 are on-pathway intermediates between Ni a -S and Ni a -C, (2) a parallel mechanism in which one Ni a -I state is an intermediate and the other is an off-pathway species, or (3) a hybrid mechanism in which one Ni a -I state is an intermediate but is in rapid equilibrium with an off-pathway state (the other Ni a -I state) (Figures S13 and S14 of the Supporting Information).…”

“…33 The Ni a -C state (green curve), which was spectrally distinct from the other resonances in the CO region, remained present at all pH values and represented the dominant state of the enzyme. In addition to the Ni a -C state, signatures of Ni a -S (red curve at ∼1949 cm −1 ) and Ni a -SR (blue curve at ∼1954 cm −1 ) were observed.…”

“…These peaks are identical to those previously reported for this enzyme at pH 7.0 (there termed Ni-L1 and Ni-L2, and herein termed Ni a -I 1 and Ni a -I 2 , respectively). 33 Additionally, a bleach feature was observed at very basic pH at 1931 cm −1 , which is present in the ground state spectrum and was assigned to the Ni r -S state. The Ni r -S state of D. vulgaris Miyazaki F [NiFe] H 2 ase was also shown to Biochemistry XXXX, XXX, XXX−XXX be photolabile.…”

“…We assign the 1940 cm −1 peak to a proton-limited fully reduced state typically termed Ni a -SR′ and the 1932 cm −1 peak to a proton-limited inactive but reduced state termed Ni r -S. Despite its small amplitude relative to the experimental noise, we consistently observe the peak at 1922 cm −1 at pH >8 and assign this state to the dark generation of one of the Ni a -I states based on its peak position, previous photochemical experiments with this enzyme, similarities in spectral shifts relative to other well-characterized H 2 ases, and its pH dependence in relation to the O 2 -tolerant Hyd1 of Escherichia coli. 33,47 An interesting and subtle effect of the pH-dependent FTIR spectra of SHI is the spectral shift of the Ni a -C resonance to a higher frequency with a decreasing pH ( Figure 2). While the observed shift is small (1.4 cm −1 ), interferometric methods are ideally suited to the detection of small frequency shifts with high accuracy.…”

Bioelectrochemical analysis of thermodynamics of the catalytic cycle and kinetics of the oxidative inactivation of oxygen-tolerant [NiFe]-hydrogenase

“…The Ni-SI:H 2 complex corresponds the Michaelis complex essentially used in the enzyme kinetics. B. L. Greene et al have also assumed the presence of H 2 -complex with Ni-SI state in the enzyme kinetic analysis [48]. Considering the Ni-SI:H 2 complex formation and a steady-state approximation, the steady-state catalytic current of the HmMBH-catalyzed H 2 oxidation (i cat ) in the presence of excess amounts of H 2 can be expressed by Eq.…”