Chemistry Reference
In-Depth Information
What type of information can we obtain from metalloprotein EPR? Examination of the EPR spectrum should
permit: (i) the identification of the type of bonding involved, based on its central hyperfine interaction, the
oxidation state of the metal ion, and possibly the type of metallo-ligand centre (for example distinguishing
between a 3Fe rather than a 4Fe cluster); (ii) quantification of the concentration of the paramagnet; (iii) structural
characterisation, which is an extension of (i), involving the identification of ligands based on ligand hyperfine
interaction of atoms in their first coordination sphere; (iv) functional characterisation, such as determining the
saturation binding of a metal ion to a specific site on the protein or using EPR spectroscopy to determine
the reduction potential of a prosthetic group in the protein. Resonances can be split into multiplet structures by the
interaction of the electron spins with nuclear spins: this gives rise to what are called hyperfine interactions. To gain
more detailed information on ligand identification and to increase resolving power, it may be necessary to apply
advanced EPR techniques such as ENDOR (electron-nuclear double resonance spectroscopy) and ESEEM
(electron spin echo envelope modulation).
Figure 6.1
a
shows the EPR spectra of horse spleen apoferritin which had been incubated with haemin.
The signal at g
¼
6 is typical of high-spin haem iron (III); a small peak at g
¼
4.3 is also present, typical of free
FIGURE 6.1
(a) EPR spectrum of horse spleen apoferritin incubated with haemin (8 haemin molecules/molecule of apoferritin) at pH 8; (b)
EPR spectrum of horse spleen apoferritin
þ
20 iron atoms/molecule of apoferritin at pH 4; (c) EPR spectrum of horse spleen apoferritin
incubated with haemin (8 haemin molecules/molecule of apoferritin) in sodium phosphate buffer, 0.1 M, pH 8, dialysed against sodium acetate
buffer, 0.1 M, pH 5.3, crystallised with ammonium sulfate and cadmium sulfate and the crystals then re-dissolved in water; (d) EPR spectrum of
horse spleen apoferritin incubated with haemin (8 molecules of haemin/molecule of apoferritin) in sodium phosphate buffer, 0.1 M, pH 8,
dialysed against sodium acetate buffer 0.1 M, pH 5.3; (e) the signal 3d was simulated as an effective S
¼
1/2 system with g-strain. The intensity
was corrected for the Boltzmann population over the three doublets of the S
¼
5/2 system assuming a zero-field splitting D
z
2cm
1
i.e., 34%
population of the middle doublet for a temperature of 16 K. Similarly, the intensity of the g
¼
6 signal from the lowest Kramers doublet of an
axial S
¼
5/2 system was corrected assuming D
z
10 cm
1
, i.e., 84% population of the m
S
¼
1/2 doublet at 16 K. For all spectra, frequency:
9.41 GHz, temperature: 16 K. (From
Carette et al., 2006
.
Copyright 2006 with permission from Elsevier.)
