202
V. Schünemann
4.4.3 Calculation of Mössbauer Parameters of Nitrophorins
Using DFT Based Methods to Proof Structural Models
of Heme Centers
The bug Rhodnius prolixus occurring in South America is not the only insect which
uses nitrophorins in order to achieve an easier blood meal. Also the bedbug cimex
lectularius uses a nitrophorin (cNP) as a NO carrying protein. Like the nitrophorins
from the Rhodnius prolixus, cNP forms a stable heme iron(III)-NO complex, where
the NO can be stored reversibly for a long period of time. Furthermore, the cNP
can bind a second NO molecule to the proximal heme cysteine when NO is present
at higher concentrations. Upon binding of the second NO molecule an S-nitrosyl
conjugate with the proximal cysteine is formed [77]. In order to confirm this proposed
reaction scheme Mössbauer spectroscopy was used to characterize the binding of the
first NO to the ferric heme unit, a situation comparable to that for the NP from
Rhodnius prolixus discussed in the previous chapter. In a second step the binding
of the second NO was investigated. During this process a reduction of the ferric
heme iron to a ferrous heme iron occurs which is well reflected in the corresponding
Mössbauer spectroscopic signatures as will be discussed below. DFT calculations
have been performed based on structural models of both scenarios [78].
Figure 4.18 shows the Mössbauer spectrum of the cNP bound to NO at pH 5.5
taken at T = 12 K with a small external field of B = 20 mT (Fig. 4.18a) and at T
= 15 K with a high external field B = 5 T (Fig. 4.18b). Spectral analyses with two
components shows that the majority component 1 has a relative intensity of 88%
and exhibits δ 1 = 0.11 mms
−1 and ΔE Q1 = 1.33 mms
−1 . The observed symmetric
doublet at low field shows in a high field of 5 T a magnetic splitting which is due
only to the external magnetic field as confirmed by the simulation representing a
diamagnetic iron species the efg of which is randomly distributed with respect to the
magnetic field direction in the frozen protein solution. Thus it can be concluded that
cNP binds one NO in the form of a diamagnetic ferric low spin heme NO just like
the nitrophorin from rhodnius prolixus as discussed in the previous chapter. When
cNP is exposed to twice the amount of NO as above and the pH is raised to pH
7.5 the Mössbauer spectrum changes drastically at low temperatures even at a low
field of 20 mT (Fig. 4.18c). Now a complex magnetic splitting is observed which
reflects a paramagnetic iron species. A spin Hamiltonian simulation using S = 1/2
gives δ 2 = 0.34 mms
−1 and ΔE Q2 = 1.42 mms
−1 . The increase of the isomer shift
reflects the reduction of the ferric low spin heme to a ferrous low spin heme center.
But ferrous low spin iron is diamagnetic, why do we observe a paramagnetic iron
species? The NO itself has S = 1/2 and binds to the diamagnetic ferrous low spin
heme creating a paramagnetic entity. In order to simulate both the Mössbauer spectra
at low (Fig. 4.18c) and high field (Fig. 4.18d) a hyperfine coupling tensor
↔
A/gnμn =
(24.4, −24.2, 12.0) T has been used. Such parameters are characteristic of a low-spin
ferrous heme iron(II)-NO complex which has been formed after the reduction of the
heme iron in the presence of high NO concentrations.
V. Schünemann
4.4.3 Calculation of Mössbauer Parameters of Nitrophorins
Using DFT Based Methods to Proof Structural Models
of Heme Centers
The bug Rhodnius prolixus occurring in South America is not the only insect which
uses nitrophorins in order to achieve an easier blood meal. Also the bedbug cimex
lectularius uses a nitrophorin (cNP) as a NO carrying protein. Like the nitrophorins
from the Rhodnius prolixus, cNP forms a stable heme iron(III)-NO complex, where
the NO can be stored reversibly for a long period of time. Furthermore, the cNP
can bind a second NO molecule to the proximal heme cysteine when NO is present
at higher concentrations. Upon binding of the second NO molecule an S-nitrosyl
conjugate with the proximal cysteine is formed [77]. In order to confirm this proposed
reaction scheme Mössbauer spectroscopy was used to characterize the binding of the
first NO to the ferric heme unit, a situation comparable to that for the NP from
Rhodnius prolixus discussed in the previous chapter. In a second step the binding
of the second NO was investigated. During this process a reduction of the ferric
heme iron to a ferrous heme iron occurs which is well reflected in the corresponding
Mössbauer spectroscopic signatures as will be discussed below. DFT calculations
have been performed based on structural models of both scenarios [78].
Figure 4.18 shows the Mössbauer spectrum of the cNP bound to NO at pH 5.5
taken at T = 12 K with a small external field of B = 20 mT (Fig. 4.18a) and at T
= 15 K with a high external field B = 5 T (Fig. 4.18b). Spectral analyses with two
components shows that the majority component 1 has a relative intensity of 88%
and exhibits δ 1 = 0.11 mms
−1 and ΔE Q1 = 1.33 mms
−1 . The observed symmetric
doublet at low field shows in a high field of 5 T a magnetic splitting which is due
only to the external magnetic field as confirmed by the simulation representing a
diamagnetic iron species the efg of which is randomly distributed with respect to the
magnetic field direction in the frozen protein solution. Thus it can be concluded that
cNP binds one NO in the form of a diamagnetic ferric low spin heme NO just like
the nitrophorin from rhodnius prolixus as discussed in the previous chapter. When
cNP is exposed to twice the amount of NO as above and the pH is raised to pH
7.5 the Mössbauer spectrum changes drastically at low temperatures even at a low
field of 20 mT (Fig. 4.18c). Now a complex magnetic splitting is observed which
reflects a paramagnetic iron species. A spin Hamiltonian simulation using S = 1/2
gives δ 2 = 0.34 mms
−1 and ΔE Q2 = 1.42 mms
−1 . The increase of the isomer shift
reflects the reduction of the ferric low spin heme to a ferrous low spin heme center.
But ferrous low spin iron is diamagnetic, why do we observe a paramagnetic iron
species? The NO itself has S = 1/2 and binds to the diamagnetic ferrous low spin
heme creating a paramagnetic entity. In order to simulate both the Mössbauer spectra
at low (Fig. 4.18c) and high field (Fig. 4.18d) a hyperfine coupling tensor
↔
A/gnμn =
(24.4, −24.2, 12.0) T has been used. Such parameters are characteristic of a low-spin
ferrous heme iron(II)-NO complex which has been formed after the reduction of the
heme iron in the presence of high NO concentrations.