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Infrared studies of the structure and mechanism of cytochrome c oxidase

Reference	BB/C51715X/1
Principal Investigator / Supervisor	Professor Peter Rich
Co-Investigators / Co-Supervisors	Dr Masayo Iwaki
Institution	University College London
Department	Genetics Evolution and Environment
Funding type	Research
Value (£)	249,431
Status	Completed
Type	Research Grant
Start date	31/08/2005
End date	30/11/2008
Duration	39 months

Abstract

Infrared spectroscopy will be used to provide atomic level information on the election and proton transfer enzymology of mammalian (bovine) and bacterial (Paracoccus denitrificans) forms of cytochrome c oxidase. Spectra will be recorded in the 4000-650 cm-1 range that covers vibrational changes of amino acids, prosthetic groups, ligands and bound water molecules. Methods have been developed in which a thin layer of enzyme in its native state is deposited on the surface of a silicon or diamond crystal. The crystal acts as the optical element in attenuated total reflectance (ATR)-FTIR spectroscopy where an IR beam is reflected internally at the prism protein interface, whilst the protein can be cycled between different forms. I have demonstrated that this method provides protein IR difference spectra of unsurpassed signal noise that allows single bond changes to be observed. Devices have been developed in my laboratory for use with the ATR-FTIR system that allow automated and simultaneous visible band difference spectra to be recorded whilst the protein is repetitively cycled between different states, for example by oxidation reduction of specific redox centres, by ligand or reactant binding removal or by conversion between different reaction cycle intermediates. This flexibility in protein manipulation has provided the means to analyse certain aspects of oxidase function that were not previously accessible. It is proposed to use these methods to analyse IR signatures of the two major catalytic intermediates, (P and F), the different conformations of the oxidised form of the enzyme, roles of specific bound water molecules and aspects of ligand-binding and redox reactions. Assignments and interpretations will be made based on band characteristics, effects of isotope-labelling and site-directed mutations, and by comparison with IR properties of related model materials. The data will be analysed in the light of published crystallographic data to provide a link from thesestatic structures to the atomic details of the dynamic catalytic mechanism of coupling of its oxygen reduction chemistry to proton and electron transfer.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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