the chromophore is protected from water molecules by the polypeptide chain. More
details can be found in Chap. 8 by Antoine and Dugourd.
It should be mentioned that spectroscopy of protein cations in vacuo is nontrivial
as one photon is seldom enough to cause dissociation. The many degrees of
freedom act, so to speak, as an internal heat bath rendering it unlikely that a
sufficient amount of energy accumulates in the dissociation mode. As a demonstration of this, Fig. 7.10 shows the time spectrum of [cytochrome c + 17H]
17+ stored in
ELISA [35]. The protein was irradiated with intense 532-nm light after 2.27 ms of
storage time in the ring; the light was the second harmonic output from an Nd:YAG
laser. Absorption is seen based on the increased yield of fragment ions measured by
the detector but at least five photons were required. Most likely the output from an
OPO would not be intense enough to cause dissociation, and wavelength scans are
therefore not easily done.
Next we consider NO binding to 4c ferric heme. Such complexes were first made
by Fornarini and co-workers [36] from ion-molecule reactions between heme ions
and NO gas. Interestingly, the stabilisation of the associative complexes was
afforded by radiative emission and not ternary collisions. This gas-phase synthesis
approach nicely eliminates the problems of reductive nitrosylation processes in
solution phase which lead to reduced iron species [36–38]. The complexes and
other Fe
III
–NO model complexes were characterised based on infrared multiphoton
photodissociation [13, 39]. Their work agreed with the formulation of the ground
state as Fe
II – NO
+ and ruled out the Fe(η
1 -ON) linkage isomer. The η
2 -NO isomer is
more than 1 eV higher in energy than the η
1 -NO isomer [13].
In recent experiments, we did spectroscopy of Fe(III)–heme
+
(NO) [14]. These
ions are weakly bound requiring only about 1 eV for dissociation, which implies
that the time scale for their dissociation is short after absorption of a visible photon
(>2 eV). An accelerator mass spectrometer was therefore used instead of the
storage ring to sample dissociation from the yield of ionic fragments produced at
each wavelength. Unlike in the storage ring where all fragmentation channels that
result in at least one neutral fragment can be measured simultaneously, each
fragmentation channel has to be measured independently at this setup. Fortunately,
however, the only photoinduced fragment of Fe(III)–heme
+
(NO) at low photon
fluxes was heme
+ (vide infra).
The action spectrum of Fe(III)–heme
+
(NO) in the Q-band region is shown in
Fig. 7.11 [14]. There, a prominent band with a maximum at 561 Æ 4 nm and
corresponding to the Q 0 band is seen. A shoulder at ~523 nm is also evident and
absorption extends up to 700 nm. For the bare Fe(III)–heme
+ ion the Q 0 band has a
maximum at 524 nm (vide supra); thus NO ligation redshifts the absorption by
37 nm. As mentioned above, the position of the Q 0 band for ferric heme(NO)
proteins with a proximal His residue was measured to lie between 549 nm and
574 nm. However, the position for most of the measured proteins lies in the range
between 560 nm and 572 nm, which is very close to that observed for the gas-phase
Fe(III)–heme
+
(NO) ion (561 nm). This again shows that the effect of the His is
limited, and thus variations at the distal site likely account for any small differences.
For example a distal histidine or water molecule in the hydrophobic protein pocket
may orient an electron lone pair towards the NO
+ ligand with a concomitant
126
J.A. Wyer and S.B. Nielsen
details can be found in Chap. 8 by Antoine and Dugourd.
It should be mentioned that spectroscopy of protein cations in vacuo is nontrivial
as one photon is seldom enough to cause dissociation. The many degrees of
freedom act, so to speak, as an internal heat bath rendering it unlikely that a
sufficient amount of energy accumulates in the dissociation mode. As a demonstration of this, Fig. 7.10 shows the time spectrum of [cytochrome c + 17H]
17+ stored in
ELISA [35]. The protein was irradiated with intense 532-nm light after 2.27 ms of
storage time in the ring; the light was the second harmonic output from an Nd:YAG
laser. Absorption is seen based on the increased yield of fragment ions measured by
the detector but at least five photons were required. Most likely the output from an
OPO would not be intense enough to cause dissociation, and wavelength scans are
therefore not easily done.
Next we consider NO binding to 4c ferric heme. Such complexes were first made
by Fornarini and co-workers [36] from ion-molecule reactions between heme ions
and NO gas. Interestingly, the stabilisation of the associative complexes was
afforded by radiative emission and not ternary collisions. This gas-phase synthesis
approach nicely eliminates the problems of reductive nitrosylation processes in
solution phase which lead to reduced iron species [36–38]. The complexes and
other Fe
III
–NO model complexes were characterised based on infrared multiphoton
photodissociation [13, 39]. Their work agreed with the formulation of the ground
state as Fe
II – NO
+ and ruled out the Fe(η
1 -ON) linkage isomer. The η
2 -NO isomer is
more than 1 eV higher in energy than the η
1 -NO isomer [13].
In recent experiments, we did spectroscopy of Fe(III)–heme
+
(NO) [14]. These
ions are weakly bound requiring only about 1 eV for dissociation, which implies
that the time scale for their dissociation is short after absorption of a visible photon
(>2 eV). An accelerator mass spectrometer was therefore used instead of the
storage ring to sample dissociation from the yield of ionic fragments produced at
each wavelength. Unlike in the storage ring where all fragmentation channels that
result in at least one neutral fragment can be measured simultaneously, each
fragmentation channel has to be measured independently at this setup. Fortunately,
however, the only photoinduced fragment of Fe(III)–heme
+
(NO) at low photon
fluxes was heme
+ (vide infra).
The action spectrum of Fe(III)–heme
+
(NO) in the Q-band region is shown in
Fig. 7.11 [14]. There, a prominent band with a maximum at 561 Æ 4 nm and
corresponding to the Q 0 band is seen. A shoulder at ~523 nm is also evident and
absorption extends up to 700 nm. For the bare Fe(III)–heme
+ ion the Q 0 band has a
maximum at 524 nm (vide supra); thus NO ligation redshifts the absorption by
37 nm. As mentioned above, the position of the Q 0 band for ferric heme(NO)
proteins with a proximal His residue was measured to lie between 549 nm and
574 nm. However, the position for most of the measured proteins lies in the range
between 560 nm and 572 nm, which is very close to that observed for the gas-phase
Fe(III)–heme
+
(NO) ion (561 nm). This again shows that the effect of the His is
limited, and thus variations at the distal site likely account for any small differences.
For example a distal histidine or water molecule in the hydrophobic protein pocket
may orient an electron lone pair towards the NO
+ ligand with a concomitant
126
J.A. Wyer and S.B. Nielsen