15N SS-NMR of Flavin Reactive Ns
A R T I C L E S
Nature’s widespread use of cofactors with broad chemical
capability makes evolutionary sense, as this strategy minimizes
the number of different cofactors which must be elaborated.
However, for a given enzyme to display only a particular
reactivity, the protein must then be able to tune and constrain
the reactivity of bound cofactors. Proteins use a variety of
devices to modulate the reactivity of bound flavins.8-15 In part,
this is achieved by virtue of the fact that the delocalized frontier
orbitals of flavins encompass several O and N functionalities
whose geometry, H-bonding, and protonation status may be
expected to strongly modulate the energy and distribution of
HOMO electron density.16-20 Thus, the energies and natures
of the flavin frontier orbitals are proposed to vary from enzyme
to enzyme, in a manner that correlates with the reactivity.20-23
Indeed, the optical, fluorescence, and Stark spectra of FMN and
FAD vary significantly depending on the protein site in which
the latter are bound.24,25 However, it is sometimes difficult to
relate such spectral differences to specific atoms or orbitals,
and the interpretation of the spectral differences may not be
unique. Flavin vibrational frequencies can be interpreted in terms
of more local effects,26-28 but possibly the most site-specific
probes available so far are EPR hyperfine couplings for the
semiquinone states29 and NMR chemical shifts for the diamag-
netic states.30-33
and hydrogen bonding at various sites, leading to valuable
insights into the polarization of the flavin ring system, its
protonation state, hydrogen bonding, and pyramidalization at
N10 and N5.30-32,34,35 However, the isotropic shifts measured
in solution represent the average of all the effects on total
electron density. Yet, many interactions involve specific orbitals,
and certain orbitals are much more important than others in
shaping flavin chemistry. One would like to obtain information
that emphasizes those.
Solid-state NMR (SS-NMR) can reveal all the elements of
the chemical shift tensor for single-crystal samples36 or measure
principal values and the corresponding eigenvectors’ orientations
relative to another vector in the molecular frame, such as a
dipole-dipole vector.37-39 Even for powder or polycrystalline
samples, SS-NMR yields all three chemical shift principal
values (CSPVs) separately. Experiments have shown that the
different CSPVs often reflect different properties, such as
protonation state vs hydrogen bonding status, for carboxyl
carbons40 and imidazole ring nitrogens.41 This is consistent with
the origins of the paramagnetic contribution to chemical shift,
whose different PVs are in theory related to different HOMO
and LUMO combinations.
Ramsey’s equation (eq 1)42 predicts that NMR chemical shifts
(δ) will reflect shielding due to ground-state electron density
(the so-called diamagnetic contribution, σii,d), as well as
deshielding due to electron orbital angular momentum (σii,p).43
NMR chemical shifts and J-couplings have been interpreted
in elegant detail in terms of π electron density, sp2/sp3 character
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-δii ) σii ) σii,d - σii,p
(1a)
(1b)
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e2
j2 + k2
σii,d
)
0
0
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|
|
2mec2
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2Lˆi
0|Lˆi|n
n
0
3
|
|
σii,p
≈
2 Σ
+
ehh
2mec
r
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(
n
(
)
En - E0
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2Lˆi
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|
r
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