A critical test of the "tunneling and coupled motion" concept in enzymatic alcohol oxidation

Abstract

The physical mechanism of C-H bond activation by enzymes is the subject of intense study, and we have tested the predictions of two competing models for C-H activation in the context of alcohol dehydrogenase. The kinetic isotope effects (KIEs) in this enzyme have previously suggested a model of quantum mechanical tunneling and coupled motion of primary (1°) and secondary (2°) hydrogens. Here we measure the 2° H/T KIEs with both H and D at the 1° position and find that the 2° KIE is significantly deflated with D-transfer, consistent with the predictions of recent Marcus-like models of H-transfer. The results suggest that the fast dynamics of H-tunneling result in a 1° isotope effect on the structure of the tunneling ready state: the trajectory of D-transfer goes through a shorter donor-acceptor distance than that of H-transfer.

Figure 1

Schematic illustration of the differences between the two models in question: a) The traditional model of tunneling and coupled motion, where the arrows represent the coupled motion of the 1° and two 2° hydrogens at the TS. As these three atoms constitute a normal mode, isotopic substitution of any of them will alter the KIE on the others. b) By the proposed model, since H’s wavefunction is more diffuse than D’s, its TRS (see figure 2) is more “dissociated”. The double-headed arrows represent the change in vibrational ZPE from ground state to TRS, which determine the 2° KIEs. c) Since D’s wavefunction is more localized than H at the TRS, its TRS is more “associated”, leading to suppression of the change in vibrational ZPE from ground state to TRS, and thus deflated 2° KIEs.

Figure 2

Marcus-like model of H-tunneling. A) The top, middle, and bottom panels show three stages of the reaction in two designated coordinates: the H position, and the positions of the heavy atoms that modulate the potential surfaces (reactant surface is blue and product surface is red) for the transferred H. In the top panel, the heavy atoms are in a position such that the zero point energy (ZPE) of the H is lower in the reactant well, so the H-wavefunction (green) is localized there. In the middle panel, the heavy atoms have rearranged to a tunneling ready state (TRS, ‡), where the ZPE for the transferred H is equal in the reactant and product wells and the H-wavefunction (including contributions from any motions coupled to the H-position) can tunnel through the barrier. The rate of reaching this tunneling ready state depends on the reaction driving force (ΔG°) and the reorganization energy (λ). In the bottom panel, the heavy atoms have rearranged further, making the ZPE of the product lower than the reactant, and thus trapping the transferred H in the product well. B) At the TRS (middle panel of A) fluctuations of the DAD affect the tunneling probability. The top panel shows a free energy surface for the designated DAD coordinate, indicating the different levels of reactant-product wavefunction overlap at different DADs. At short enough DAD, the ZPE of the transferred particle may be above the barrier, but this leads to very small 1° KIEs and does not appear to be the case for ADH. The middle panel shows the Boltzmann probability distribution of the system being at any given DAD (magenta), along with the tunneling probabilities of H and D as a function of DAD (orange and purple, respectively). The bottom panel shows the product of the Boltzmann factor and the tunneling probability for each particle, yielding the probability of a reactive trajectory as a function of DAD. Panel B illustrates that the reactive trajectories for H and D go through different average DADs, constituting an isotope effect on TRS structure. In ADH, the difference in average DAD for hydride transfer vs. deuteride transfer, which was estimated as 0.2 Å, leads to differences in 2° KIEs when the transferred isotope is different.

Scheme 1

The reaction catalyzed by ADH (using benzyl alcohol as an alternative substrate). R= adenine diphosphate ribosyl-.