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Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

ABSTRACT: Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H2O and D2O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

SUBMITTER: Adamczyk K

PROVIDER: S-EPMC5588449 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

Adamczyk Katrin K Simpson Niall N Greetham Gregory M GM Gumiero Andrea A Walsh Martin A MA Towrie Michael M Parker Anthony W AW Hunt Neil T NT

Chemical science 20141022 1

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we ...[more]

PMID: 28936306

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Project description:Proteins, enzymes, and other biological molecules undergo structural dynamics as an intrinsic part of their biological functions. While many biological processes occur on the millisecond, second, and even longer time scales, the fundamental structural dynamics that eventually give rise to such processes occur on much faster time scales. Many decades ago, chemical kineticists focused on the inverse of the reaction rate constant as the important time scale for a chemical reaction. However, through transition state theory and a vast amount of experimental evidence, we now know that the key events in a chemical reaction can involve structural fluctuations that take a system of reactants to its transition state, the crossing of a barrier, and the eventual relaxation to product states. Such dynamics occur on very fast time scales. Today researchers would like to investigate the fast structural fluctuations of biological molecules to gain an understanding of how biological processes proceed from simple structural changes in biomolecules to the final, complex biological function. The study of the fast structural dynamics of biological molecules requires experiments that operate on the appropriate time scales, and in this Account, we discuss the application of ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy to the study of protein dynamics. The 2D IR vibrational echo experiment is akin to 2D NMR, but it operates on time scales many orders of magnitude faster. In the experiments, a particular vibrational oscillator serves as a vibrational dynamics probe. As the structure of the protein evolves in time, the structural changes are manifested as time-dependent changes in the frequency of the vibrational dynamics probe. The 2D IR vibrational echo experiments can track the vibrational frequency evolution, which we then relate to the time evolution of the protein structure. In particular, we measured protein substate interconversion for mutants of myoglobin using 2D IR chemical exchange spectroscopy and observed well-defined substate interconversion on a sub-100 ps time scale. In another study, we investigated the influence of binding five different substrates to the enzyme cytochrome P450(cam). The various substrates affect the enzyme dynamics differently, and the observed dynamics are correlated with the enzyme's selectivity of hydroxylation of the substrates and with the substrate binding affinity.

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Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

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Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

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