78
M. Seto et al.
2.3.2 Examples of Frontier Science, Especially Biological
Application
Element (isotope) selectivity is very advantageous when the function and local structure of a specific atom in a complex compound are of interest. Resonant Raman spectroscopy provides valuable information on the vibrational properties of samples, such
as metalloprotein samples. Furthermore, it has been used effectively. However, the
selection rule sometimes hampers the observation of vibrational modes. In contrast,
NRIS method gives all modes that involve motion of the resonant nuclei. Therefore,
it provides the opportunity to study the ligation and oxidation state of a specific
site. Many studies have been conducted to determine the vibrational amplitudes
and frequencies for specific atoms in a complex biological macromolecule without
selection rules using the isotope selectivity (for example [54–76]). In particular, this
method has been used to study specific sites in large molecules, such as metalloprotein samples, involving thousands of other atoms. The investigation of the structure
around the specific site of interest is conducted by comparing the obtained phonon
energy spectrum with the vibrational spectrum obtained from DFT calculations under
the assumption of the presumable structural model. This method is effective for
samples that cannot be crystallized, such as the intermediates in the catalytic cycles
of enzymes. Furthermore, the structural characterization of the reactive Fe(IV) = O
intermediate in the catalytic cycles of a mononuclear non-heme iron (NHFe) enzyme
(the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae) was
studied (Fig. 2.13) [72]. The intermediate reacts through an initial hydrogen-atom
abstraction step and performs subsequent halogenation of the native substrate or
hydroxylation of nonnative substrates, Therefore, the revelation of its local structure
and mechanism is quite essential and important. In this study, it was indicated that
the orientation of the Fe(IV) = O intermediate depends on the substrate, presenting
specific frontier molecular orbitals responsible for hydrogen-atom abstraction that
can selectively lead to halogenation or hydroxylation. Moreover, NRVS was used
in studying the catalytic mechanism of hydrogenases, which catalyze the reversible
conversion of molecular hydrogen to protons and electrons. Understanding the mechanism is quite significant because it leads to the development of clean energy sources
in producing hydrogen. NRVS was applied to [FeFe] hydrogenase variant lacking the
amine proton shuttle, which is stabilizing a putative hydride state [75]. [FeFe] hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen
with extremely high efficiency. NRVS spectra clearly showed the bending modes of
the terminal Fe–H species that is consistent with the widely accepted models of the
catalytic cycle.
The high brilliance of SR allows the PDOS even for small samples to be measured.
For example, measurements under extreme conditions in which the accessible sample
space is severely limited are possible. The PDOS under high pressures using a
diamond anvil cell (DAC), where the sample size is typically less than 1 mm
2 ,
is measured to study the core of the earth [77–79]. Additionally, many important
studies using the features of NRIS spectroscopy have been conducted, for example,
M. Seto et al.
2.3.2 Examples of Frontier Science, Especially Biological
Application
Element (isotope) selectivity is very advantageous when the function and local structure of a specific atom in a complex compound are of interest. Resonant Raman spectroscopy provides valuable information on the vibrational properties of samples, such
as metalloprotein samples. Furthermore, it has been used effectively. However, the
selection rule sometimes hampers the observation of vibrational modes. In contrast,
NRIS method gives all modes that involve motion of the resonant nuclei. Therefore,
it provides the opportunity to study the ligation and oxidation state of a specific
site. Many studies have been conducted to determine the vibrational amplitudes
and frequencies for specific atoms in a complex biological macromolecule without
selection rules using the isotope selectivity (for example [54–76]). In particular, this
method has been used to study specific sites in large molecules, such as metalloprotein samples, involving thousands of other atoms. The investigation of the structure
around the specific site of interest is conducted by comparing the obtained phonon
energy spectrum with the vibrational spectrum obtained from DFT calculations under
the assumption of the presumable structural model. This method is effective for
samples that cannot be crystallized, such as the intermediates in the catalytic cycles
of enzymes. Furthermore, the structural characterization of the reactive Fe(IV) = O
intermediate in the catalytic cycles of a mononuclear non-heme iron (NHFe) enzyme
(the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae) was
studied (Fig. 2.13) [72]. The intermediate reacts through an initial hydrogen-atom
abstraction step and performs subsequent halogenation of the native substrate or
hydroxylation of nonnative substrates, Therefore, the revelation of its local structure
and mechanism is quite essential and important. In this study, it was indicated that
the orientation of the Fe(IV) = O intermediate depends on the substrate, presenting
specific frontier molecular orbitals responsible for hydrogen-atom abstraction that
can selectively lead to halogenation or hydroxylation. Moreover, NRVS was used
in studying the catalytic mechanism of hydrogenases, which catalyze the reversible
conversion of molecular hydrogen to protons and electrons. Understanding the mechanism is quite significant because it leads to the development of clean energy sources
in producing hydrogen. NRVS was applied to [FeFe] hydrogenase variant lacking the
amine proton shuttle, which is stabilizing a putative hydride state [75]. [FeFe] hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen
with extremely high efficiency. NRVS spectra clearly showed the bending modes of
the terminal Fe–H species that is consistent with the widely accepted models of the
catalytic cycle.
The high brilliance of SR allows the PDOS even for small samples to be measured.
For example, measurements under extreme conditions in which the accessible sample
space is severely limited are possible. The PDOS under high pressures using a
diamond anvil cell (DAC), where the sample size is typically less than 1 mm
2 ,
is measured to study the core of the earth [77–79]. Additionally, many important
studies using the features of NRIS spectroscopy have been conducted, for example,