ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P4. Fluorescence Correlation Spectroscopy in the Study of Fast Biological
Electron Transfer Reactions
A. Andreoni a , A. W. J. W. Tepper a , S. Kuznetsova a , L. C. Tabares a , T. J. Aartsma b ,
G. W. Canters a
a
Leiden Institute of Chemistry, Leiden University, Einsteinweg, 55, 2333CC, Leiden, Netherlands
e-mail: aandreoni@chem.leidenuniv.nl
b
Leiden Institute of Physics, Leiden University, Niels Bohrweg, 2, 2333CA, Leiden, Netherlands
Azurin is a 14 kDa blue-copper protein that serves as electron carrier in cells. Oxidised Azurin can donate an
electron to different partners including reduced Azurin. By using chemically cross-linked Cu(I)/Cu(II)-Azurin
dimers it is possible to measure the electron self-exchange between the monomers[1]. In the present work a
novel method to measure this electron transfer process is presented. Oxidized Azurin has a strong absorption
band at 628nm that disappears upon reduction. By using a covalently attached fluorescent dye it was possible to
translate this change in absorption to a change in fluorescence by means of Forster Resonance Energy Transfer
(FRET): while Azurin is reduced no FRET occurs but after oxidation part of the energy absorbed by the dye was
transfer to the Cu(II) resulting in a decrease of the emitted fluorescence. This effect allowed the detection of the
Cu redox state in Azurin dimers at single molecule level by Fluorescence Correlation Spectroscopy. Experiments
were performed on Cy5 labelled Cu- or Zn-Azurin dimers. While for the redox inactive Zn-dimers the
autocorrelation curve fit well to a simple diffusion model an extra parameter was necessary to fit the Cu-dimers
data. A model to explain this different behaviour that includes the electron self-exchange between the Cu centres
was developed and the kinetic data obtained from it are presented. The results show that this method is suitable
for the investigation of electron transfer processes in proteins.
Figure: Illustration of the FRET effect behaviour while electron transfer within the dimer occurs
References:
[1] van Amsterdam IM, Ubbink M, Einsle O, Messerschmidt A, Merli A, Cavazzini D, Rossi GL, Canters GW,
Nat Struct Biol.; 9(1):48-52 (Jan 2002)
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