200
V. Schünemann
Since in the case of NP2-Hm the g-tensor is quite anisotropic it turns out that
also the spin expectation value
− →
S = (S x , S y , S z ) is quite anisotropic. In
addition, also the hyperfine coupling tensor tensor
↔
A of ferric low spin centers is quite
anisotropic. The components of
↔
A can be determined by simulation of Mössbauer
spectra obtained at different magnetic fields at low temperature (e.g. ~4.2 K) in order
to make sure that the spectra are not blurred by electronic relaxation effects. The
simulations than should fit the different spectra equally well, a task which had to be
done by manual simulations in the early times of Mössbauer spectroscopy. Nowadays
this can be performed with the help of a simultaneous fitting of several spectra, a
feature that can be implemented in e.g. VINDA [29]. This has been done by means of
the simulations displayed in Fig. 4.16d–f. It should be noted that it is allowed only to
vary the parameter which has been varied in the experiment, in this case the external
field, all other parameters should be the same for all simulations. Such a simulation
requires quite a high number of parameters like the three components of
↔
A, the
asymmetry parameter η, the sign of ΔE Q as well as its absolute value. The latter can
be also determined by Mössbauer experiments at temperatures where the electronic
spin relaxation rate is fast in the Mössbauer time window and only a quadrupole
splitting is observed. This value can be taken as an input for the simulation. However,
one should be aware that the quadrupole splitting can be temperature dependent when
excited electronic states are populated. In some cases it might be even necessary to
introduce the Euler angles between the main axis system of the g-tensor and those
of the electric field gradient and the magnetic hyperfine tensor.
There is also a possibility to calculate relatively straightforward theoretically
values of the hyperfine tensor components
↔
A from experimentally obtained g-values
for ferric low spin species. Oosterhuis and Lang [68] developed analytical equations
which describe at least to our experience the experimentally observed components
of the
↔
A tensor components of ferric low spin hemes quite well.
4.4.2 Investigation of Vibrational Properties of Nitrophorins
In this chapter we will discuss the investigation of ligand binding to nitrophorins
using NIS. The application of this technique to heme proteins started with several
studies of the O 2 storage protein myoglobin [70–76]. One may ask the question what
information can be gained by using a complicated synchrotron technique in contrast
to laboratory based Raman- and or Infrared-Spectroscopy. The advantage of using
the
57 Fe nucleus as a nuclear probe also for the study of the vibrational dynamics
is that there are no optical selection rules other than that the iron has to move with
respect to the direction of the incoming synchrotron beam. In addition, Raman and
IR spectroscopy do detect a whole variety of vibrations of the protein environment
which may mask the relevant modes one is hunting for especially when Fe ligand
modes are concerned. Figure 4.17 shows the NIS raw data of NP2–NO, NP2–CN
V. Schünemann
Since in the case of NP2-Hm the g-tensor is quite anisotropic it turns out that
also the spin expectation value
− →
S = (S x , S y , S z ) is quite anisotropic. In
addition, also the hyperfine coupling tensor tensor
↔
A of ferric low spin centers is quite
anisotropic. The components of
↔
A can be determined by simulation of Mössbauer
spectra obtained at different magnetic fields at low temperature (e.g. ~4.2 K) in order
to make sure that the spectra are not blurred by electronic relaxation effects. The
simulations than should fit the different spectra equally well, a task which had to be
done by manual simulations in the early times of Mössbauer spectroscopy. Nowadays
this can be performed with the help of a simultaneous fitting of several spectra, a
feature that can be implemented in e.g. VINDA [29]. This has been done by means of
the simulations displayed in Fig. 4.16d–f. It should be noted that it is allowed only to
vary the parameter which has been varied in the experiment, in this case the external
field, all other parameters should be the same for all simulations. Such a simulation
requires quite a high number of parameters like the three components of
↔
A, the
asymmetry parameter η, the sign of ΔE Q as well as its absolute value. The latter can
be also determined by Mössbauer experiments at temperatures where the electronic
spin relaxation rate is fast in the Mössbauer time window and only a quadrupole
splitting is observed. This value can be taken as an input for the simulation. However,
one should be aware that the quadrupole splitting can be temperature dependent when
excited electronic states are populated. In some cases it might be even necessary to
introduce the Euler angles between the main axis system of the g-tensor and those
of the electric field gradient and the magnetic hyperfine tensor.
There is also a possibility to calculate relatively straightforward theoretically
values of the hyperfine tensor components
↔
A from experimentally obtained g-values
for ferric low spin species. Oosterhuis and Lang [68] developed analytical equations
which describe at least to our experience the experimentally observed components
of the
↔
A tensor components of ferric low spin hemes quite well.
4.4.2 Investigation of Vibrational Properties of Nitrophorins
In this chapter we will discuss the investigation of ligand binding to nitrophorins
using NIS. The application of this technique to heme proteins started with several
studies of the O 2 storage protein myoglobin [70–76]. One may ask the question what
information can be gained by using a complicated synchrotron technique in contrast
to laboratory based Raman- and or Infrared-Spectroscopy. The advantage of using
the
57 Fe nucleus as a nuclear probe also for the study of the vibrational dynamics
is that there are no optical selection rules other than that the iron has to move with
respect to the direction of the incoming synchrotron beam. In addition, Raman and
IR spectroscopy do detect a whole variety of vibrations of the protein environment
which may mask the relevant modes one is hunting for especially when Fe ligand
modes are concerned. Figure 4.17 shows the NIS raw data of NP2–NO, NP2–CN