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Spectroscopic and kinetic properties of the horseradish peroxidase mutant T171S

journal contribution
posted on 2023-06-07, 22:52 authored by Barry D Howes, Nigel Brissett, Wendy A Doyle, Andrew T Smith, Giulietta Smulevich
Studies on horseradish peroxidase C and other haem peroxidases have been carried out on selected mutants in the distal haem cavity providing insight into the functional importance of the distal residues. Recent work has demonstrated that proximal structural features can also exert an important influence in determining the electronic structure of the haem pocket. To extend our understanding of the significance of proximal characteristics in regulating haem properties the proximal Thr171Ser mutant has been constructed. Thr171 is an important linking residue between the structural proximal Ca2+ ion and the proximal haem ligand, in particular the methyl group of Thr171 interdigitates with other proximal residues in the core of the enzyme. Although the mutation induces no significant changes to the functional properties of the enzyme, electronic absorption and resonance Raman spectroscopy reveal that it has a highly selective affect on the reduced state of the enzyme, effectively stabilizing it, whilst the electronic properties of the Fe(III) state unchanged and essentially identical to those of the native protein. This results in a significant change in the Fe2+/Fe3+ redox potential of the mutant. It is concluded that the unusual properties of the Thr171Ser mutant reflect the loss of a structural restraint in the proximal haem pocket that allows 'slippage' of the proximal haem ligand, but only in the reduced state. This is a remarkably subtle and specific effect that appears to increase the flexibility of the reduced state of the mutant compared to that of the wild-type protein.

History

Publication status

  • Published

Journal

FEBS Journal

ISSN

1742-464X

Publisher

Wiley-Blackwell

Issue

21

Volume

272

Page range

5514-5521

Pages

8.0

Department affiliated with

  • Biochemistry Publications

Notes

ATS directed mutagenesis, spectroscopic and stopped flow studies carried out at Sussex, manuscript drafted by ATS as joint corresponding author, resonance Raman done in collaboration with Florence group.

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2012-02-06

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