Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy

Abstract

Enzymes move on a variety of length and timescales. While much is known about large structural fluctuations which impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme catalyzed reactions. This letter reports frequency fluctuations of azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the frequency-frequency correlation function when the azide is bound to the NAD+ ternary complex. This oscillation disappears when the reduced cofactor is added, indicating that the oscillating contributions most likely come from the charged nicotinamide ring. These oscillatory motions may be relevant to donor-acceptor distance sampling of the catalyzed hydride transfer, and therefore may give future insights into the dynamic behavior involved in enzyme catalysis.

Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase

“…Some researchers have tried to demonstrate, specifically in enzyme-catalysed hydrogen transfer reactions, that quantum mechanical hydrogen tunnelling associated with enzymatic X-H bond cleavage links to protein dynamics for achieving optimal enzyme catalysis. 33,37,[44][45][46] These studies are based on the temperature dependence of the kinetic isotope effect (KIE) method that has been used in recent years to study the nature of H-transfer enzymatic reactions and can be a powerful tool for understanding the role of dynamics in enzyme catalysis.…”

“…80 QM/MM free energy simulations on the recombinant CbFDH enzyme yielded a free energy profile in agreement with the experimental data and it is very similar to the one obtained from PsFDH. 86 Recently, two-dimensional infrared spectroscopy measurements on the ternary complex of FDH with cofactor and the azide as the transition state analogue, carried out by Kohen and co-workers in CbFDH, 46 revealed structural fluctuations on the picosecond to femtosecond timescale, which could be relevant to catalysis. The measurements of the frequency-frequency correlation function for the azide complexed to FDH with NAD + showed underdamped oscillatory frequency fluctuations on the picosecond timescale.…”

Protein Mass Effects on Formate Dehydrogenase

“…43 A transition state analog of the reactive ternary complex, (FDH-azide-NAD + ) is used to experimentally probe the active site dynamics of FDH. 43-46 …”

Compressive sampling methods applied to 2D IR spectroscopy

“…We present 2D IR data collected in our lab measuring the antisymmetric stretch of the azide anion bound to formate dehydrogenase in a ternary complex with NAD + to determine the frequency-frequency correlation function (FFCF). 37 The full data set required 8-10 days of data acquisition because of the high density of waiting times and the extensive averaging necessary for the measurement. For each 2D IR spectrum, we scan τ 1 from 0 to 4 ps, taking 24 fs steps in the rotating frame and use a four-pulse phase cycle to isolate the signal.…”

“…where we use a sum of exponential functions that includes damped cosines to model the oscillatory features in the decay. We have described the origin of the oscillatory features elsewhere, 37 and here we focus on the ability of compressive sampling to resolve these oscillations and return the correct frequencies, time constants, and relative amplitude parameters. Increasing compression results in increases in the errors on the parameters of both the high-frequency (blue) and low-frequency (red) oscillatory terms with the most substantial effect occurring between 4× and 6× compression.…”