By introducing more than one spin probe to a protein, the coupling
between the spin probes can be measured to study conformational
changes of the protein.
Research direction: heme proteins
Heme proteins such as myoglobin, hemoglobin, and cytochrome are
globular proteins that have, as cofactors, hemes, which are planar
molecules with central iron atoms (Figure 16.21). The central iron
atom can have an EPR signal depending upon the state of ligation.
There are four nitrogens in the heme plane, which always serve as
ligands and two out-of-plane coordination sites (Figure 16.21).
Myoglobin in the deoxy form has Fe^2 + with two axial histidine
ligands. Oxygen can bind to the sixth coordination position and the
iron remains Fe^2 +. Alternatively, with otherligands the oxidation
state becomes Fe^3 +. As the coordination of the iron atom changes,
the electronic structure also changes. For example, the changes of
the electronic states correspond to shifts in the allowed transition
energies, resulting in a distinctive optical spectrum for each state
(Figure 16.22).
These relationships between coordination of the metal and the
electronic state can be understood in terms of ligand or crystal field
theory. Whereas an understanding of the EPR spectra requires a
detailed understanding of the properties of individual transition
metals, the effect of different coordinations can be explained on a
qualitative level. Consider as an example the iron of heme. The
EPR signal of the iron is dependent upon the properties of the
five and six electrons in the d orbitals for Fe^3 +and
Fe^2 +respectively. The energies of the individual d
orbitals can be describedby considering the differ-
ent types of electronic interaction usinga hierarchical
approach. In ligand field theory, the nature of the
ligands is not considered, only the relative location
and number. For heme, the iron can be considered
to be coordinated by four equivalent nitrogens in
the heme plane with different ligands along the
heme normal, providinga tetragonal field.
The ferric state of iron has five electrons in the
outer d orbitals. These five electrons will fill the five
d orbitals to minimize the energy of the system.
The d orbitals can be energetically grouped into the
three orbitals, dxy, dyz, and dzx, which are more
sensitive to the x–ycoordination (these are termed
the t 2 gorbitals) and the two orbitals, dxx, which are
more sensitive to the axial coordination (these are
CHAPTER 16 MAGNETIC RESONANCE 367
Figure 16.21Coordination
of the Fe in the heme
group of myoglobin to
(top) nitrogen atoms in
plane and (bottom) amino
acid residues.500 550 600OxyhemoglobinDeoxyhemoglobinAbsorbanceWavelength (nm)
Figure 16.22The optical spectra of
different states of myoglobin.