2 Materials and Methods
3.2 Sulfite reductase from Archaeoglobus fulgidus
3.2.4 EPR spectroscopy
3.2.4.2 Oxidized sulfite reductase
Sulfite reductase as isolated was reacted with potassium ferricyanide under exclusion of dioxygen. This resulted in rather unexpected EPR spectral changes (Figure 3.25). In the low field part there were only 2 absorption shaped lines at g=17.5 and 9.7. Also the mid field part was dominated by a single component with g=6.65 and g=5.1.
Although a quantitation was not performed, it was clearly visible that the intensity of the signals was much higher than in sulfite reductase as isolated, even more if a correction factor
for the concentration was introduced (conditions for the comparison of the normalized spectra: microwave power, 0.6 mW; modulation amplitude, 1 mT; temperature, 10 K).
Interestingly the relative intensity of the S=9/2 signals compared to the S=5/2 signals decreased from the state as isolated to the oxidized state.
40 60 80 100 120 140 160 180
A
Magnetic Field [mT]
15.0 11.3 9.07 7.57 6.49 5.68 5.06 4.55 4.14 3.80 g- Value
30 40 50 60 70 80 90
Magnetic Field [mT]
18.0 15.0 12.9 11.3 10.1 9.07 8.25 7.57
B g- Value
Figure 3.25: A EPR spectrum of sulfite reductase from A. fulgidus. B Low-field spectrum of sulfite reductase. EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 0.6 mW; modulation amplitude, 1 mT; temperature, 10 K.
At high magnetic field (Figure 3.26) there were still resonances from two species present:
species I with gx=1.978, gy=2.007, gz=2.03, species II with gx=1.958, gy=2.007, gz=2.073. The relative contribution of the two changed. In the spectrum of the enzyme as isolated their relative contribution was approximately equal. After the reaction with ferricyanide the relative contributions were about 10:1. This was consistent with the assumption of species II being a [4Fe-4S] cluster of the ferredoxin type. It was partly reduced in the enzyme as isolated and was then almost completely oxidized to the S=0 [4Fe-4S]2+ form.
300 320 340 360 380
Magnetic Field [mT]
2.21 2.14 2.09 2.03 1.98 1.93 1.88 1.83 1.79
g- Value
Figure 3.26: Upper trace: high-field spectrum of sulfite reductase from A. fulgidus. Lower trace:
simulation. EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH
7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 0.6 mW; modulation amplitude, 1 mT; temperature, 10 K.
At low and medium magnetic field the EPR spectrum of oxidized sulfite reductase (Figure 3.27) was simulated as a sum of one species with S=9/2 and four species with S=5/2. In comparison with the enzyme as isolated the signal of adventitiously bound Fe(III) was reduced.
40 60 80 100 120 140 160 180
g- Value
Magnetic Field [mT]
15.0 11.3 9.07 7.57 6.49 5.68 5.06 4.55 4.14 3.80
30 40 50 60 70 80 90
A B
Magnetic Field [mT]
g- Value
18.0 15.0 12.9 11.3 10.1 9.07 8.25 7.57
Figure 3.27: A Upper trace: EPR spectrum of sulfite reductase from A. fulgidus. Lower trace:
simulation. B Upper trace: low-field spectrum of oxidized sulfite reductase. Lower trace: simulation.
EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 2 mW;
modulation amplitude, 1 mT; temperature, 6 K.
The simulation of the low and mid field part of the spectrum resulted in a rather accurate fit.
The low-field part (Figure 3.28A) could be simulated with a single S=9/2 system with E/D=0.153. The line at g=17.5 originated from the | ± 1/2 > doublet, the | ± 3/2 > doublet was responsible for the line at g=9.7.
The dominating S=5/2 component (Figure 3.28B) was simulated with E/D=0.036. One might have argued about the E/D value of this component in view of the offset of 27 mT between the observed and simulated position of the absorption shaped line. It was not possible with any E/D to get the absorption shaped as well as the derivative shaped line to coincide with the measured data. Usually one would have claimed that this was due to deviations from the weak-field first order perturbation approach as a result of small zero-field splitting. However, the simulation program WEPR diagonalized the spin Hamiltonian of the S=5/2 system so this couldn’t be the reason for the deviation. In addition, even with the low D between 2.4 and 4.9 (Table 3.8) the weak-field limit seemed appropriate. The reason for this deviation was most likely mixing of the S=5/2 with other spin states (Burdinsky et al., 2001). This typical
signature of a ‘spin-admixed’ state was due to the spin-orbit coupling of ground state with excited states in this case most likely the first excited state with S=3/2.
There were also minor contributions from components with E/D=0.057, E/D=0.013 and E/D=0.0265.
30 40 50 60 70 80 90
Magnetic Field [mT]
90 105 120 135 150 165
Magnetic Field [mT]
18.7 16.1 14.1 12.6 11.3 10.3 9.44 8.72 8.10 7.57
A B
e
c d
b a g-Value
7.32 6.68 6.14 5.68 5.29 4.95 4.64 4.38 4.14
g- Value
Figure 3.28: Contribution of the simulated sub spectra to the EPR-spectrum of sulfite reductase from A. fulgidus. A Upper trace: low-field part of the EPR spectrum of sulfite reductase. Lower trace:
simulation with S=9/2 and E/D=0.153. B Part of the spectrum with S=5/2 signals with a E/D=0.057 b E/D=0.013 c E/D=0.0265 d E/D=0.036. e EPR-spectrum of sulfite reductase. EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 2 mW; modulation amplitude, 1 mT;
temperature, 6 K.
3.2.4.2.1 Temperature dependence and zero field splitting
The EPR spectrum of oxidized sulfite reductase showed the following behavior with increasing temperature:
(i) the signal at g=17.5 (38 mT) decreased,
(ii) the signal at g=9.7 (69 mT) displayed its maximum intensity at 8K, (iii) the S=5/2 signals from g=5.2 to g=7.3 (90 to 135 mT) decreased.
The ferredoxin-like signals (gx=1.978, gy=2.007, gz=2.03) had their maximum intensity at 10K.
40 60 80 100 120 140 160 180
A B
Magnetic Field [mT]
15.0 11.3 9.07 7.57 6.49 5.68 5.06 4.55 4.14 3.80
12 K
300 310 320 330 340 350 360 370 380
Magnetic Field [mT]
2.21 2.14 2.09 2.03 1.98 1.93 1.88 1.83 1.79
g- Value
Figure 3.29: Temperature dependence of the EPR spectrum of sulfite reductase from A. fulgidus.
Lower trace 5.5 K, middle trace 8 K, upper trace 12 K. A Low-field spectrum B high-field spectrum.
EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 0.6 mW;
modulation amplitude, 1 mT.
The sign of the zero-field splitting parameter D of oxidized sulfite reductase needed to be positive as in sulfite reductase as isolated. The single S=9/2 species with the | ± 1/2 > doublet at g=17.5 (38 mT) and the | ± 3/2 > doublet at g=9.7 (79 mT) showed the expected behavior (Figure 3.31). The | ± 1/2 > doublet decreased in intensity with increasing temperature. The
| ± 3/2 > doublet first increased in intensity with temperature as the doublet got populated and then decreased with a further increase in temperature.
0.08 0.10 0.12 0.14 0.16 0.18 0.20
-0.2
0.08 0.10 0.12 0.14 0.16 0.18 0.20
-40
Figure 3.30: Determination of the zero-field splitting D for the A S=9/2 and B S=5/2 system of sulfite reductase from A. fulgidus. The baseline corrected intensities were fitted to the corresponding curie corrected Boltzmann distribution. A The lines at g=17.5 (▲) and 9.7 (■) were fitted to a | ± 1/2 > and
| ± 3/2 > system with E/D=0.153. B The lines at g= 6.65 (▲), 5.12 (●) and (■) were fitted to a | ± 1/2 >
system with E/D=0.036.
As in sulfite reductase as isolated the fitting of the temperature dependence of the signal from the | ± 3/2 > doublet was much more precise than the one from the | ± 1/2 > doublet (Table
3.8). The value of 4.1 ± 0.4 was in agreement with the value for sulfite reductase as isolated.
In the case of oxidized sulfite reductase it was also possible to determine the zero field splitting of the S=5/2 system. This was due to the fact of the reduced number of components contributing to the EPR spectrum. The value of around 3.5 was rather low for a siroheme (Pierik & Hagen, 1991).
Spin g-Value E/D Doublet χ2 D ±D
9/2 17.5 0.153 1/2 0.00062 1.0 12.4
9/2 9.7 0.153 3/2 0.00438 4.1 0.4
5/2 6.65 0.036 1/2 0.364 3.4 1.0 5/2 5.12 0.036 1/2 1.18 10.1 7.8 5/2 5.12 0.036 1/2 0.242 3.8 1.1 Table 3.8: Zero-field splitting parameters of oxidized sulfite reductase from A. fulgidus. The Error in D does not include the experimental error.
The temperature dependence of the EPR spectrum for the S=9/2 system was simulated using the parameters in Table 3.9.
For the S=9/2 system both the lines from the | ± 1/2 > and the | ± 3/2 > doublet were observed.
The zero-field splitting influenced their relative intensity. In addition these lines were observed at different temperatures. The temperature behavior was also dependent on the zero-field splitting. The value of D from depopulation (Table 3.8) did not reflect the correct relative intensities for the | ± 1/2 > and the | ± 3/2 > doublet. The simulation calculated the correct transition probabilities for the | ± 1/2 > and the | ± 3/2 > doublet. Therefore a D value was used that fitted best with the relative intensities for the | ± 1/2 > and the | ± 3/2 > doublet.
It had to be noted that it was difficult to simulate the temperature dependence as the simulated spectra (Figure 3.31) were normalized with respect to the total second integral. This integral could be treated as a Curie system. In order to determine its value at various temperatures its value at 0 K had to be known. With the used cryostat it was only possible to go as low as 4.5 K. On the other hand the EPR spectrum of the S=9/2 system (Figure 3.31 traces a-e) was highly temperature dependent. Small deviations from the selected temperature resulted in large deviations from the expected intensity and even larger deviations from the correct zero-field splitting.
30 40 50 60 70 80 90
sim
sim
sim
sim
sim e
d
c
b
a
Magnetic Field [mT]
30 40 50 60 70 80 90
Magnetic Field [mT]
18.7 16.1 14.1 12.6 11.3 10.3 9.44 8.72 8.10 7.57
g- Value
Figure 3.31: Temperature dependence of the resonances at low field of sulfite reductase from A.
fulgidus. a sulfite reductase at 5.5K. b sulfite reductase at 6K. c Sulfite reductase at 8K. d sulfite reductase at 10K. e sulfite reductase at 12K. EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.378 GHz; microwave power, 0.6 mW; modulation amplitude, 1 mT.
The analysis of the relative contribution of the simulated sub-spectra reflected the qualitative impression of the EPR spectra of sulfite reductase oxidized versus enzyme as isolated. For the simulation a single S=9/2 component and one dominating S=5/2 component were used.
Spin Rhombicity Zero-field splitting [cm-1] Linewidth [MHz] Contribution [%]
9/2 0.153 2.4 250 100
5/2 0.013 3.5 125 2.0
5/2 0.0265 3.5 125 11.9
5/2 0.036 3.5 125 84.1
5/2 0.057 3.5 125 2.0
5/2 0.33 3.5 80 0.1
Table 3.9: Parameters for the simulation of EPR-spectrum of oxidized sulfite reductase from A.
fulgidus using the program WEPR (Neese, 1995)
3.2.4.2.2 Power saturation studies
The dependence of the EPR signal intensity on the microwave power P was studied at 6K.
The saturation behavior (half-saturation, P1/2) of the S=9/2 and the S=5/2 system were rather similar. The half saturation power was around 1-2 mW for both major components. This was rather surprising compared to the saturation behavior of sulfite reductase as isolated.
-3 -2 -1 0 1 2 3
Figure 3.32: Power saturation study on sulfite reductase. A Low field resonances at g=17.5 (■) and g=9.7 (○). B Mid field resonances at g=7.3 (■), g=6.6 (●), g=6.1 (○), g=5.7 (U), g=5.4 (x), g=5.2 (▲) and g=5.0 (□). EPR conditions: 15 mg ml-1 oxidized sulfite reductase in 100 mM potassium phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency, 9.38 GHz; microwave power, 0.002 - 160 mW; modulation amplitude, 1 mT; temperature, 6 K.
The details of the half-saturation power determination were taken from Figure 3.32 and listed in Table 3.10. Table 3.10: Power saturation study on oxidized sulfite reductase from A. fulgidus at 6 K.
Summarizing the results above: in oxidized sulfite reductase there was one S=9/2 species with E/D= 0.153. The determination of the zero-field splitting parameter D was with the method of thermal depopulation only reliable for the | ± 3/2 > doublet but did not agree with the value
from the simulation. The good simulation and the steep intensity dependence on D suggested a more reliable D determination by simulation than by thermal depopulation. The zero-field splitting for the E/D=0.153 component was D = 4.1 ± 0.4 cm-1 (2.4 cm-1 by simulation). The value of the half-saturation power of around 2 mW was rather reliable but the small signals and the use of peak height instead of peak area had to be kept in mind.
In addition, there were four S=5/2 species with E/D= 0.057, 0.036, 0.0265 and 0.013. The zero-field splitting for the E/D=0.036 component was self-consistent with an average D = 3.5 ± 1 cm-1. The power saturation studies for these signals were reliable and P1/2 was in the range of 0.7-2 mW for the different components (~2 mW for the major component).