The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase

Niall Simpson, Katrin Adamczyk, Gordon Robert Hithell, Daniel Shaw, Gregory M. Greetham, Michael Towrie, Anthony William A.W. Parker, Neil Hunt

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D<inf>2</inf>O and H<inf>2</inf>O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism. This journal is

LanguageEnglish
Pages163-179
Number of pages17
JournalFaraday Discussions
Volume177
Early online date21 Jan 2015
DOIs
Publication statusPublished - 1 Apr 2015

Fingerprint

Horseradish Peroxidase
Heme
enzymes
Molecules
dynamic structural analysis
Water
Structural dynamics
Substrates
water
molecules
Enzymes
acids
probes
nitric oxide
Stretching
Infrared spectroscopy
Nitric Oxide
infrared spectroscopy
Pumps
pumps

Keywords

  • infrared spectroscopy
  • substrate binding
  • haem ligand
  • water molecules

Cite this

Simpson, Niall ; Adamczyk, Katrin ; Hithell, Gordon Robert ; Shaw, Daniel ; Greetham, Gregory M. ; Towrie, Michael ; Parker, Anthony William A.W. ; Hunt, Neil. / The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase. In: Faraday Discussions. 2015 ; Vol. 177. pp. 163-179.
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abstract = "Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism. This journal is",
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Simpson, N, Adamczyk, K, Hithell, GR, Shaw, D, Greetham, GM, Towrie, M, Parker, AWAW & Hunt, N 2015, 'The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase' Faraday Discussions, vol. 177, pp. 163-179. https://doi.org/10.1039/C4FD00161C

The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase. / Simpson, Niall; Adamczyk, Katrin; Hithell, Gordon Robert; Shaw, Daniel; Greetham, Gregory M.; Towrie, Michael; Parker, Anthony William A.W.; Hunt, Neil.

In: Faraday Discussions, Vol. 177, 01.04.2015, p. 163-179.

Research output: Contribution to journalArticle

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AU - Towrie, Michael

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