6.3 Conclusion and Outlook
7.1.3 HYSCORE Spectra of Isotope Labeled Samples
68 7.1 The Oxidized Ready State Ni-B and Unready State Ni-A
Determination of Proton, Deuteron, and Nitrogen Hyperfine Couplings 69
2
2
H
D
[MHz]ν
H O
ν ν
12 14 16
2
12 4
−2
−4
−6 6
0 2 4 6
14 16
νN
+ +
+
2
1 1
H
D
[MHz]ν
ν [MHz] ν
ν
[MHz]
12 14 16
2
12 4
6
−2
−4
−6 0 2 4 6
14 16
N
ν ν
+ +
+
Figure 7.5: HYSCORE spectra of the hydrogenase from D. vulgaris Miyazaki F at g2in H2O (Top) and D2O (Bottom). Arrows indicate spectrometer artifacts related to the pulse programmer clock which occur at
3.9, 7.9, 11.8, 15.7, .. MHz. Experimental conditions: (Top) Ni-B: 70 %, B0= 323.4 mT, 4.2 K, 9.77 GHz,π 2 16 ns,π 24 ns,τ 120 ns, (Bottom) Ni-B: 85 %, B0= 323.2 mT, 4 K, 9.77 GHZ,π 2 16 ns,π 24 ns,τ1 120 ns,τ2 160 ns.
Ni-B nitrogen couplings. Along the diagonal the three zero-field transitions of the nitrogen coupling emerged due to non-ideality of the pulses. Off-diagonal correlation transitions betweenν0ν ν and νdq with varying intensities were detected; (ν0νdq) at (0.6,3.69) MHz, (ν νdq) at (1.35,3.69) MHz and (1.25,4.1) MHz, (ν νdq) at (1.88,3.69) MHz and (1.65,4.1) MHz.
Furthermore, very weak signals stretched from about (20.3, 7.4) MHz to (22.0, 5.6) MHz perpen-dicular and symmetrically spaced with respect to the main diagonal (data not shown) which may be at-tributed toβ-CH2protons of the cysteine residues. The hfc’s for these protons have a large isotropic con-tribution (cf. [125, 157]) which leads to a large splitting from the diagonal. Due to the small anisotropy, peak intensities are very low for these protons (see Section 3.3.1).
Initial hf parameters for the simulation of the ridge stretching from (14.9, 13.5) MHz - (15.7, 12.6) MHz have been determined from the spectra of the hydrogenase in H2O according to the method devel-oped by P¨oppl et al. [110]: aiso 1.9 0.4 MHz and adip -3.9 0.4 MHz or aiso -1.6 0.4 MHz and adip 2.4 0.4 MHz. As a starting point for the simulations it was assumed that this proton
70 7.1 The Oxidized Ready State Ni-B and Unready State Ni-A
Table 7.2: The hf couplings of the hydrogenase from D. vulgaris Miyazaki F as obtained by simulation of the HYSCORE spectra of the enzyme in H2O. The direction cosines of the hyperfine tensor are given relative to the crystal structure [23]. Further simulation parameters: gi 2.333, 2.163, 2.010; Wi
1.2 mT, 0.7 mT, 0.6 mT. Comparison of experimental spectra with simulations while systematically varying one parameter resulted in the following estimated margins of error: ∆a
01 MHz for the exchangeable proton and∆a 003 MHz for the 14N hfc,∆ e2qQ h
003 MHz,∆η 002, angles
15 20 for the exchangeable proton and
10 for14N.
exchangeable H Ni-B
1 2 3
aiso [MHz] 1.9
adipi [MHz] 4.4 2.9 -7.3
Ai [MHz] 6.3 4.8 -5.5
lai 0.26 0.57 0.78
lbi 0.95 -0.29 -0.10
lci 0.17 0.77 -0.61
e2qQ h [MHz] –
η –
lai lbi
lci
N(ε)-His88 Ni-B
1 2 3
-1.95
0.25 0.15 -0.40 -1.65 -1.75 -2.30 0.37 0.85 0.36 -0.13 -0.34 0.93 0.92 -0.39 -0.01
1.96 0.39
-0.59 0.76 0.27 0.48 0.06 0.88 0.65 0.65 -0.39
N(ε)-His88 Ni-A
1 2 3
-1.48
0.20 0.15 -0.35 -1.28 -1.33 -1.83 -0.22 0.82 0.52 0.20 -0.49 0.85 0.95 0.29 -0.06
1.90 0.35
0.80 0.49 0.35 0.02 -0.56 0.83 0.60 -0.67 -0.43
is bound at the small bridging ligand like it was proposed by Bleijlevens et al. [150]. Thus, initial values for the Euler angles relating the g-tensor orientation to the hf tensor orientation were calculated from the position of the bridging ligand in the X-ray structure of the oxidized enzyme and using the direction cosines of the g-tensor principal axes of the Ni-A and Ni-B state [79]. A hydroxyl group, which was assumed being a ligand to the nickel site, in most cases is not bound linearly with respect to the metal-O bonds. Accordingly, the Euler angles were varied over a wide range during the simulation procedure.
The variation of the direction cosines induced only slight changes to the form of the ridge, accordingly the magnitudes of the initially obtained direction cosines are more erroneous than those which were determined by the following analysis. Iteratively, the parameters of the simulation of the exchangeable proton were used for the refinement of the simulation of the exchangeable deuteron and vice-versa (see below) in order to get a consistent set of orientation parameters. The ridge at 14.2 MHz in H2O buffer was simulated with best agreement with the shape of the ridge in the experimental spectrum with one set of hfc parameters. The resulting hfc values are given in Table 7.2 and the simulated spectrum is displayed in Figure 7.7.
Due to the presence of at least two paramagnetic nuclei coupling with the unpaired electron spin
Determination of Proton, Deuteron, and Nitrogen Hyperfine Couplings 71
2
1
[MHz] ν
ν [MHz]
a b
2 4 6
−2
−4
−6 0 2 4 6
Figure 7.6: Simulation of the HYSCORE spectrum of the oxidized hydrogenase from D. vulgaris Miyazaki F at g2. (a) The simulated 2D HYSCORE spectrum of the14N couplings for the Ni-A (red) and Ni-B (green) state are depicted. They are assigned to an adjacent histidine residue, His88. For the presentation of the simulated spectra a ratio Ni-A:Ni-B = 3:7 was considered. The contours of the experimental spectrum (depicted in Figure 7.5) are underlayed in grey. (b) Skyline projections of the experimental (—) and simulated ( ) HYSCORE spectra. The simulation is the sum of 0.3 Ni-A and 0.7 Ni-B. Simulation parameters: see Table 7.2 and B0= 323,4 mT, 9.77 GHz,τ 120 ns.
Figure 7.7: Simulation of the HYSCORE spectrum of the the exchangeable proton of the oxidized hydrogenase from D. vulgaris Miyazaki F at g2(green). The contours of the experimental spectrum (shown in Figure 7.5) are underlayed in grey. The simulation param-eters used are displayed in Table 7.2. The spec-trum has been calculated with B0= 323.4 mT, 9.77 GHz,τ 120 ns. By takingτ=210 ns, the splitting of the ridge could be reproduced.
1
2
ν
[MHz]ν
[MHz]
12 14 16
12 14 16
νH
+
one expects that combination peaks of notable intensities emerge. This effect would alter the spectrum compared to these of only one nucleus. Such ridges are quite intense, especially if e g a deuterium nucleus (I=1) is coupled to a14N nucleus (I=1) [158]. In order to understand the HYSCORE spectra of the H/D exchanged sample which will be discussed below one first has to know the full set of parameters
72 7.1 The Oxidized Ready State Ni-B and Unready State Ni-A of the14N nucleus and could then extract the hyperfine and quadrupole parameters for deuterium by simulation.
The14N quadrupole couplings have been studied earlier by ESEEM studies of the Ni-A state of the hydrogenase from D. gigas in solution [159] and extensively by single crystal ESEEM and HYSCORE spectroscopy on the Ni-A and Ni-B state of the hydrogenase from D. vulgaris Miyazaki F [148].
According to the characteristic values of e2qQ h and η the nucleus has been assigned to a weakly coupled imidazole N [159] rather than to a backbone N or other nitrogen. In hydrogenases a highly conserved histidine residue is found, namely His88, which is located in the close proximity of the [NiFe]-cluster.7.1h This residue has been identified to be responsible for the coupling by a combination of site directed mutagenesis and spectroscopic experiments on a related system, the regulatory hydro-genase from R. eutropha [131]. Results of DFT calculations on 2-methyl-imidazole and ethanthiol as a model strongly suggest that the N(ε)-H of His88 forms a hydrogen bond with the sulfur atom of Cys549 [130, 131]. This residue is in the direction of the g3 axis and thus is suspected to carry a part of the spin density [81, 124, 142, 160]. However, the hyperfine parameters for this 14N have not been determined so far.
Initial values for e2qQ h andηfor Ni-B: e2qQ h =1.98 MHz,η= 0.37 and for Ni-A e2qQ h =1.90 MHz,η= 0.34 were taken [130]. These were varied in order to obtain better agreement of the simu-lated spectra with respect to the experiment. The resulting deviation of the final simulation parameters were within the given error. For the simulation it was initially assumed that the principal axes of the quadrupole tensor axes are collinear with the one of the hf tensor which is justifiable according to Ashby et al. [161]: The X-ray structure revealed that the probable orientation of the lone pair or the N-H bond at the N(ε), which is approximately the bisector of the imino CNC bond, should fall into the direction of the dipolar axis [23]. In a further step of the simulation procedure the direction cosines of the principal axes of the quadrupole coupling tensor were varied, too. The results of the spectral simulation for the N(ε) of His88 for the Ni-A and the Ni-B state are presented in Table 7.2 and in Figure 7.6.
Deuteron HYSCORE Spectra. Upon solvent exchange (85 % Ni-B and 15 % Ni-A)7 1gthe separated ridge at 13.8 MHz vanished completely. At the same time at the 2H Larmor frequency new signals emerged (Figure 7.5, 7.8). These signals now can clearly be attributed to the exchangeable proton found by X-band cw-EPR spectroscopy which is suspected to be part of a ligand to the [NiFe] site in the first ligand sphere due to the coupling and the pronounced anisotropy [150]. The other features of the spectrum, i.e. the weakly coupled nitrogen and the relatively isotropic couplings in the proton region, were maintained to first approximation ( e.g. (ν νdq) at (1.15,4.0) MHz and (ν νdq) at (1.7,4.0) MHz)). Surprisingly, the H/D exchanged sample exhibited 14N couplings slightly different from those in the spectra of the hydrogenase in H2O: The double quantum peaks showed a larger splitting of the correlation peaks of (νsq1 νdq), (νsq2νdq), correlations associated with v are shifted to slightly lower
7.1hIn the hydrogenase from D. vulgaris Miyazaki F the N(ε) is located in a distance of 5.10 ˚A from the Ni center.
Determination of Proton, Deuteron, and Nitrogen Hyperfine Couplings 73
2
1
1 3
1
[MHz]ν
ν [MHz]
g
2 4 6
2 4 6
g
ν [MHz]
2 4 6
Figure 7.8: HYSCORE spectra of the hydrogenase from D. vulgaris Miyazaki F at g1and g3in D2O.
(g1) B0= 299.5 mT, 4 K, 9.77 GHz,π 2 16 ns,π 24 ns,τ1 176 ns,τ2 200, (g3) B0= 338.9 mT, 4 K, 9.77 GHZ,π 2 16 ns,π 24 ns,τ1 120 ns,τ2 176 ns,τ2 200 ns, accumulation times 3-8 h, each.
frequencies and the ridges are broadened.
In order to interpret the spectra of the enzyme in D2O, first the14N signals were simulated and then the contributions of the deuteron were analyzed. The simulation parameters for the hyperfine and quadrupole interaction of the N(ε) of His88 previous simulation thus were tentatively changed until a fairly good agreement with the experimental spectrum was obtained (Table 7.3 and Figure 7.9).
The proton hf couplings obtained in the precedent simulation of the spectra have been scaled down by the factor gH gH to hfc values for a deuterium nucleus. The orientation and hyperfine parameters have been refined iteratively until the orientation dependence of the hfc has been reproduced satisfac-torily. However, the relative intensities of the double quantum transitions as well as of the combination peaks were comparatively low which turned out to be disadvantageous for unambiguously deriving the hfc parameters. The coupling to a deuterium nucleus (I=1) has a certain quadrupole contribution even though the quadrupole moment is about an order of magnitude smaller than that of a nitrogen nucleus.
Such a contribution has been found in case of a hydroxo ligand at the Mo center of sulfite oxidase and of the dimethyl sulfoxide (DMSO) reductase [162,163] and has also been observed in the case of a deuter-ated tetrapyridine-copper(II) complex [164]. The correlation peaks of the HYSCORE spectra showed a clear splitting which has been simulated with an e2qQ h of 0.25 MHz7.1i [162, 163]. However, in a spectrum at g2obtained by division of the HYSCORE time traces (envelope division) [165] of the D.
vulgaris Miyazaki F [NiFe]-hydrogenase no such splitting with e2qQ H 0 2 MHz could be observed within the given linewidth. Analogously, 2H-ENDOR spectra of the reoxidized form of the D. gigas
7.1iThese spectra have been recorded at Ku-band frequency (16.5 GHz) in order to meet the exact cancellation condition νD A 2 which produces sharp lines.
74 7.1 The Oxidized Ready State Ni-B and Unready State Ni-A
Table 7.3: The hf couplings of the hydrogenase from D. vulgaris Miyazaki F as obtained by simulation of the HYSCORE spectra of the D2O exchanged the enzyme. The hfc’s for deuteron are a factor gN 1H gN 2H 6514 smaller than for protons. Further simulation parameters: gi 2.333, 2.163, 2.010; Wi 1.2 mT, 0.7 mT, 0.6 mT. (errors: see Table 7.2) and∆a 005 MHz for the2H hfc coupling.
exchangeable D Ni-B
1 2 3
aiso [MHz] 0.28
adipi [MHz] 0.67 0.45 -1.12 Ai [MHz] 0.95 0.73 -1.40
lai 0.26 0.57 0.78
lbi 0.95 -0.29 -0.10
lci 0.17 0.77 -0.61
e2qQ h [MHz] –
η –
lai lbi
lci
N(ε)-His88 Ni-B
1 2 3
-1.85
0.23 0.15 -0.38 -1.62 -1.70 -2.23 0.37 0.85 0.36 -0.13 -0.34 0.93 0.92 -0.39 -0.01
1.90 0.52
-0.59 0.76 0.27 0.48 0.06 0.88 0.65 0.65 -0.39
hydrogenase did not reveal any splitting of the signals due to quadrupole interaction [37]. Accordingly, no quadrupole splitting has been considered in the simulations. After several refinement cycles the hfc parameters for the exchangeable deuteron have been determined to the values given in Table 7.3. The simulated spectra are depicted in Figure 7.9.