heme group covalently attached to the cytochrome c protein. The maximum of the
band for the native structure in solution is very close to the value reported for the
gas-phase protein (see inset in Fig. 8.6). However, when the protein is denatured in
solution, the Soret band redshifts (see inset in Fig. 8.6), which demonstrates how
sensitive the Soret band is to the local environment. The present result constitutes a
benchmark for simulating the absorption of prosthetic chromophores within a
protein environment without having to take into account solvent molecules.
8.6
Spectroscopy of Peptide Radical Ions
Radical centres present in peptides and proteins draw considerable interest for two
major reasons: they play critical roles as intermediates in a variety of biological
electron-transfer processes, and they provide a wealth of fragmentation pathways
that are relevant to protein sequencing. Whereas the optical properties of closedshell tryptophan in the environment of a protein is well documented, direct
observations of radicals—and particularly radical cations—have been ailed by
their high, environment-modulating reactivity.
Electron detachment from the doubly deprotonated TrpValValValVal peptide
([M-2H]
2À ) leads to the formation of an indolyl radical (see inset in Fig. 8.7a). The
photogenerated radical was stable and could be isolated for several tens of
milliseconds. In a two-colour experiment (see Scheme 8.2), the long-lived radical
ions were isolated and, after isolation, irradiated with a second tuneable visible
laser pulse (MS
3 experiments) [21].
The fragmentation spectrum of the radical was systematically recorded as a
function of the wavelength of the second laser in the 430–590 nm range. The
resulting experimental photofragmentation spectrum is shown in Fig. 8.7a. It
displays a single absorption band centred at 473 nm. This band is in the visible
300
400
500
600
300
400
500
600
C...
...N
N •
C...
...N
HN
nm
+ •
[TrpValValValVal]-•
Radical Trp
[AcGly 3 TrpNH 2 ]+•
Radical CaƟonTrp
a
b
Fig. 8.7 Photofragmentation yield as a function of the laser wavelength for the gas phase (a)
TrpValValValVal oxidised anions [M-2H]
À• and (b) [AcGly 3 TrpNH 2 ]
+• radical cations. The
indole residue is in a neutral radical form in (a) and in a radical cation form in (b)
8 UV–Visible Absorption Spectroscopy of Protein Ions
149
band for the native structure in solution is very close to the value reported for the
gas-phase protein (see inset in Fig. 8.6). However, when the protein is denatured in
solution, the Soret band redshifts (see inset in Fig. 8.6), which demonstrates how
sensitive the Soret band is to the local environment. The present result constitutes a
benchmark for simulating the absorption of prosthetic chromophores within a
protein environment without having to take into account solvent molecules.
8.6
Spectroscopy of Peptide Radical Ions
Radical centres present in peptides and proteins draw considerable interest for two
major reasons: they play critical roles as intermediates in a variety of biological
electron-transfer processes, and they provide a wealth of fragmentation pathways
that are relevant to protein sequencing. Whereas the optical properties of closedshell tryptophan in the environment of a protein is well documented, direct
observations of radicals—and particularly radical cations—have been ailed by
their high, environment-modulating reactivity.
Electron detachment from the doubly deprotonated TrpValValValVal peptide
([M-2H]
2À ) leads to the formation of an indolyl radical (see inset in Fig. 8.7a). The
photogenerated radical was stable and could be isolated for several tens of
milliseconds. In a two-colour experiment (see Scheme 8.2), the long-lived radical
ions were isolated and, after isolation, irradiated with a second tuneable visible
laser pulse (MS
3 experiments) [21].
The fragmentation spectrum of the radical was systematically recorded as a
function of the wavelength of the second laser in the 430–590 nm range. The
resulting experimental photofragmentation spectrum is shown in Fig. 8.7a. It
displays a single absorption band centred at 473 nm. This band is in the visible
300
400
500
600
300
400
500
600
C...
...N
N •
C...
...N
HN
nm
+ •
[TrpValValValVal]-•
Radical Trp
[AcGly 3 TrpNH 2 ]+•
Radical CaƟonTrp
a
b
Fig. 8.7 Photofragmentation yield as a function of the laser wavelength for the gas phase (a)
TrpValValValVal oxidised anions [M-2H]
À• and (b) [AcGly 3 TrpNH 2 ]
+• radical cations. The
indole residue is in a neutral radical form in (a) and in a radical cation form in (b)
8 UV–Visible Absorption Spectroscopy of Protein Ions
149