7.2 The Catalytically Active Intermediate Ni-C
7.2.1 EPR Spectroscopic Characterization of the Ni-C State
Determination of EPR Line-Widths of Ni-C in H2O and D2O. Exposition of D. vulgaris Miyazaki F hydrogenase samples to a pure hydrogen gas atmosphere for 2-3 h yielded the Ni-C state. The spin concentration of Ni-C in D. vulgaris Miyazaki F hydrogenase was about 30-50 % of that found for the oxidized states. The respective EPR spectra at X- and Q-band frequencies of this species in H2O/H2
and D2O/D2 are shown in Figure 7.12. The generation of the deuterated hydrogenase samples in the Ni-C state had taken up to three times longer incubation times. A narrowing of the EPR linewidths at all canonical orientations was observed upon solvent exchange to D2O and subsequent reduction of the sample with D2(Figure 7.12, Table 7.4). Besides unresolved ligand hyperfine splittings the line widths of the Ni-C EPR signal arise essentially from g-strain effects.
The spin concentration of Ni-C in the D. vulgaris Miyazaki F hydrogenase corresponds to spin concentrations reported before for hydrogenases from other organisms [37, 61]. The line narrowing upon H/D exchange is in the same range like reported for other hydrogenases [37, 47, 60]. The line-widths of Ni-C are comparable in size to those of the Ni-B state of this enzyme and also compare well with values determined for the D. gigas hydrogenase [74]. In both states the broadening was most pronounced at g1 which points to microheterogeneities in the structure of the active site especially in that direction. The g-tensor axes g1and g2are, however, interchanged upon conversion of Ni-A/Ni-B
82 7.2 The Catalytically Active Intermediate Ni-C
Table 7.4: g-tensor principal values of the Ni-C state of the hydrogenase from D. vulgaris Miyazaki F determined at X- and Q-band frequencies by simulation of the EPR spectra of Figure 7.12 using an in-house simulation program [121]. (∆g(X-band) 0003,∆g(Q-band) 00006,∆W
0.2 mT).
g-values linewidth [MHz]
Ni-C H2O X-band 2.198 2.146 2.011 1.9 2.0 1.8 D2O X-band 2.199 2.146 2.011 1.5 1.7 1.5 H2O Q-band 2.1980 2.1462 2.0112 3.3 1.7 (0.2)
to Ni-C.
’Split’ Ni-C. Lowering the temperature yielded the complex EPR spectrum of ’split’ Ni-C; the max-imum signal intensity of the virtually ’unsplit’ Ni-C signal was obtained at 40-50 K, see Figure 7.12.
The EPR lines split into two for g1 and g2 or into more lines at g3. The splitting of the signals at the three canonical orientations was determined from the spectra at X- (and Q-band) with 5.6 (5.1), 13.3 (12.5), 3.3/10.3 (9.1) mT. The g1feature with the smallest splitting broadened out first when the temper-ature was raised again whereas g2 with the largest splitting broadened out last. This is expected as the broadening occurs when the magnitude of the splitting, given in frequency units, is comparable to the spin lattice relaxation rate 1/T1at a given temperature. The condition for this is 1/T1 ∆gβB h, where
∆B is the splitting in magnetic field units [72]. Accurate information about the magnitude of exchange and dipolar splittings have been obtained before by measurements at multiple frequencies and complex simulations on the D. gigas hydrogenase [74, 134, 173, 174] which shows a highly similar splitting pattern.7.2b With the presence of this splitting the application of advanced EPR methods like ENDOR or HYSCORE spectroscopy is prohibited due to strongly enhanced relaxation rates by the magnetic coupling with the fast relaxing iron-sulfur clusters [49, 74, 175].
pH Dependence of Ni-C. It was attempted to find experimental conditions for the generation of
’unsplit’ Ni-C which is obtained when the [4Fe-4S]-clusters are oxidized. As the midpoint potential of the conversion from the Ni-C to Ni-Si state exhibits a larger pH dependence with respect to the iron-sulfur clusters [8, 44, 49, 52] it was assumed that the fraction of the reduced [4Fe-4S]-clusters would decrease upon lowering the pH and thus would yield pure unsplit Ni-C. The resulting EPR signal shapes of the Ni-C species prepared at different pH values were not influenced7.2cwhich is in line with
7.2bIt has been shown earlier that these two hydrogenases are closely related concerning the structure and spatial arrangement of the cofactors as well as their general redox behavior [23, 46]. However, subtle differences in the exact midpoint potentials are observed which may be caused by the altered protein environments of the cofactors of the respective hydrogenase.
7.2cThis was tested in the range of pH 5.0 - 8.0 in steps of 0.5 pH units. At pH values lower 5.5 - 5 the protein started to denature quite rapidly even at low concentrations.
Determination of Proton, Deuteron, and Nitrogen Hyperfine Couplings 83
Figure 7.12: X-band and Q-band EPR spec-tra of the Ni-C state of the hydrogenase from D. vulgaris Miyazaki F. X-band: (a) in H2O, (b) in D2O, and (c) low temperature ’split’
Ni-C in H2O. Q-band: (d) in H2O and (e) low temperature ’split’ form. The simula-tions ( ) were obtained using an in-house simulation program [121] with the parame-ters given in Table 7.4. Experimental con-ditions: (a,b) T = 50 K, 9.44 GHz, 1 mW microwave power, 0.28 mT modulation am-plitude, (c) T = 5 K, 9.59 GHz, 1 mW mi-crowave power, 0.28 mT mod. amp., (d) T = 50 K, 33.994 GHz, 0.42 mW, 1.1 mT mod.
amp., (e) T = 5.9 K, 33.995 GHz, 0.1 mW, 1.1 mT mod. amplitude.
2 2.1
H O D O
a b
c
d
e
2.2
g−value
Q−band X−band
2
2
observations on the [NiFeSe]-hydrogenase from Methanococcus voltae [176] and on hydrogenase from A. vinosum reported only recently [177]. However, the integrals of the EPR signal varied with the pH, as expected according to the observations of Zorin et al. [178]. At higher pH values larger EPR integrals were obtained. The relative concentration of Ni-C at lower pH values was increased with respect to pH 8 (pH 6 : pH 7 : pH 8 = 1 : 0.75 : 0.7) but all the EPR spectra recorded at low temperature showed fully
’split’ Ni-C signals,7.2dsee Figure 7.13.
Generation of ’Unsplit’ Ni-C. Samples of the D. vulgaris Miyazaki F hydrogenase containing an increased fraction of up to 50-70 % of the ’unsplit’ Ni-C form only could be generated successfully by purging the Ni-C sample for 15 to 30 minutes with argon as described before [74]. Thereby the Ni-C concentration was lowered with respect to the initial concentration. Upon longer equilibration times with argon, the Ni-C signal intensity decreased further, then first the [3Fe-4S]
signal, and subsequently the Ni-A signal reemerged. In previous electrochemical studies midpoint potentials of the nickel center of the hydrogenase from D. vulgaris Miyazaki [46] have been reported being higher than those observed for the D. gigas hydrogenase [44, 52]. Thus, most probable the midpoint potentials of the other metal cofactors of the D. vulgaris Miyazaki F hydrogenase are less separated than in the D. gigas enzyme
7.2dIn order to set a defined potential several redox dyes (a compilation is given in [179]) also was used. However, a sample with ’unsplit’ Ni-C with sufficiently high concentrations was not obtained this way.
84 7.2 The Catalytically Active Intermediate Ni-C
7 K 40 K
Figure 7.13: X-band EPR spectra of Ni-C of the D. vulgaris Miyazaki F hydrogenase buffered at different pH values at 40 K (Left) and 7 K (Right). The signal intensities were normalized to the initial enzyme concentration. Experimental conditions: 9.44 GHz, 1 mW microwave power, 100 kHz mod.
frequency, 1 mT mod. amplitude.
(cf. Figure 2.3). However, these potentials have not been determined so far. It has been reported for the D. gigas hydrogenase that it was possible to generate pure ’unsplit’ Ni-C by purging the reduced sample with argon gas [74].
In principle, it is of interest to produce single crystalline samples of ’unsplit’ Ni-C. These samples would yield in a very elegant manner spatial information about paramagnetic nuclei coupled to the electron spin carrying center by means of site and orientation selected advanced EPR techniques, such as ENDOR or ESEEM spectroscopy. Such studies have been undertaken successfully with oxidized single crystals of the D. vulgaris Miyazaki F hydrogenase [125, 130, 148]. However, especially for the Ni-C state, these investigations would require perfect crystals in a much larger size than available in order to obtain a reasonable signal to noise ratio.
In Figure 7.14 a representative EPR spectrum of a mixed Ni-C and Ni-L state is depicted which is partially ’split’. Obviously, the whole range of both species, Ni-C and Ni-L, is superposed by the complex EPR spectrum of a spin-coupled iron-sulfur cluster.7.2e These species may also contribute ENDOR or HYSCORE spectra in the respective temperature range where the spin coupling is observed.
This will be discussed in the respective sections.
7.2ecf. [74], the g-values o the iron-sulfur cluster of the hydrogenase from D. gigas are 1.860,1.915, 2.137.
Determination of Proton, Deuteron, and Nitrogen Hyperfine Couplings 85
Figure 7.14: Example of an X-band EPR spectrum of the hydrogenase from D. vulgaris Miyazaki F with a mixture of the partially ’un-split’ Ni-C (60 %, magenta) and Ni-L state (40
%, blue) obtained after purging the reduced sample with argon. For further measurements the respective pure states have been produced.
The broad signal with the apparent g-values of 1.90, 1.72 stems from the spin coupled iron-sulfur clusters [173]. Experimental conditions:
T = 6K, 9.67 GHz, microwave power 1mW,
mod. frequ. 100 kHz, mod. amp. 1 mT. 3 2.5 2 1.5
g−value