J. Phys. Chem. 1994,98, 467-473
461
Electron Spin Echo Envelope Modulation Studies of Water Bound to Tetracyanonickelate(111) John McCracken' and Steven Friedenberg Department of Chemistry. Michigan State University, East Lansing, Michigan 48824 Received: August 20, 1993; I n Final Form: October 15, 19930
Electron spin echo envelope modulation studies aimed a t characterizing the superhyperfine coupling between protons of axially bound water molecules and the nickel ion in Nirt1(CN)4(H20)2- were carried out. X-band, two-pulse ESEEM experiments showed that this coupling is characterized by a large anisotropic component resulting in 0.1-1.6-MHz shifts of the Y, + vb proton sum combination peak from twice the Larmor frequency. A means of reducing these data into a field profile constructed by plotting these frequency shifts as a function of magnetic field strength across the EPR absorption envelope is presented. Computer simulations of typical field profiles are shown to be sensitive to the effective Ni-H dipole-dipole distance, the orientation of the principal axis of the hyperfine coupling tensor with respect to the g-tensor, and to a lesser extent, the Fermi contact or scalar hyperfine coupling term. For water coordinated to Ni1rr(CN)4(H20)2-, analysis of the field profile yields an effective dipole-dipole distance of 2.4 A, an angle describing the orientation of All for the proton superhyperfine coupling tensor with respect to the g3 axis of 1 2 O , and a scalar coupling 1 4 MHz. These results are discussed in the context of recent ENDOR studies of the nickel site in Ni-Fe hydrogenases.
Introduction
Electron paramagnetic resonance (EPR) methods have played an important role in characterizing the structure and biological function of the nickel cofactor of Ni-Fe hydrogenases. Continuous wave (cw)EPR studies of the oxidized, inactive form of the enzyme performed at 77 K show an intense absorption that arises from a composite of two nickel species. Both signals are indicative of either Ni(II1) or Ni(1) and are characterized by rhombic g-tensors. The dominant species, Ni-A, has principal g-values of 2.31, 2.26, and 2.02, and the second, more weakly absorbingspecies, Ni-B, has principalg-values of 2.33,2.16, and 2.02.lJ Confirmation that both of these signals arise from nickel was provided by 6lNi isotopic substitution studies of hydrogenases isolated from Methanobacterium thermautotrophicum and Desuljbvibrio gigas. In these investigations, pronounced hyperfine splittings of 27 G, consistent with coupling to an Z = 3/2 nucleus, were found for the high-field spectral feature at g = 2.02.394 The measured g-values for these signals are similar to those found for model complexes with tetragonally elongated octahedral coordination geometries where much of the unpaired electron spin density resides in the nickel 3d9 orbital.5 The nickel ion is most likely Ni(II1) since both Ni-A and Ni-B forms are observed for the oxidized state of the enzyme. When the enzyme is activated by reduction with hydrogen, the Ni-A and Ni-B EPR signals are rapidly lost and a new signal known as Ni-C appears.' Ni-C is also characterized by a rhombic g-tensor with principal values of 2.19,2.15, and 2.02 and shows similar, -27 G, 61Nihyperfine splittingsof theg = 2.02 feature when isotopically labeled enzyme is studied. In contrast to the Ni-A and Ni-B forms of the protein where appreciable 6INi hyperfine splitting was also observed at the two lower field EPR features, little coupling is observed at g = 2.19 or 2.15 for the Ni-C form.' These cw-EPR results are characteristic of either Ni(II1) or Ni(1) where the coordination geometry has been altered by reduction of the enzyme, but may still be approximated by a tetragonally distorted octahedron.5 The parallel rise in amplitude of the Ni-C EPR signal and enzyme catalytic activity with reduction have led to the proposal that the nickel site comprises at least part of the substrate binding site.' Pulsed EPR experimentsusing the electron spin echo envelope modulation (ESEEM) technique have been carried out on the Author to whom correspondence should bc addreaaed.
*Abstract published in Aduance ACS Absrracrs, December 15, 1993.
0022-3654/94/2098-0467S04.50/0
Ni-Fe hydrogenase from D. gigas to gain more detailed information on the structure and catalytic function of the nickel site. A comparison of ESEEM data for enzyme in aqueous buffer with those obtained for enzyme exchanged against 2H20 buffer showed that the nickel site was inaccessible to solvent unless the protein was in its active Ni-C form.6 The 2H-ESEEM signal observed for Ni-C in the exchanged samples consisted of a single, broad peakcenteredat the deuterium Larmor frequency. Because this spectral feature may contain contributions from the exchangeable protons of the nickel ligands as well as those from solvent molecules and exchangeable protons on the protein backbone close to the metal, a more detailed analysis of the *HESEEM data is precluded. Subsequent Q-band electron nuclear double-resonance(ENDOR) studies of this enzyme resolved two pairs of exchangeableproton resonances with hyperfine couplings of 4.4 and 16.8 MHz.' In a more recent Q-band ENDOR study of a Ni-Fe hydrogenase from Thiocapsa roseopersicina, exchangeable proton resonances with hyperfine couplings of approximately 20 MHz were observed.8 In this paper we report on ESEEM studies of the proton hyperfine couplings of water ligands axially coordinated to NiI11(CN)4(H20)2-. This study represents a first step in the development of a systematic means by which advanced EPR methods can be used to detect and distinguish HzO, OH-, and H- ligands bound to paramagnetic transition ions in a variety of chemical systems and to help provide a foundation upon which ESEEM and ENDOR results from studies of the nickel site in hydrogenasescan be interpreted. The tetracyanonickelatesystem was chosen for this study because it is a good 'magnetic model" for the Ni-C species, with gL 2 gll and principal g-values of 2.20, 2.20, and 2.01. Also, 61Ni isotopic substitution studies show a hyperfinesplitting of the cw-EPR spectrum that is similar to that found for reduced enzyme.9J0 Two pulse ESEEM methods that ultilize the proton v, up or sum combination peak for the detection of ligand hyperfine couplings characterized by large anisotropiccomponents were used in this study.11-14In addition, a convenient graphical approach for enhancing the analysis of these data for electron spin systems dominated by g-tensor anisotropy and/or hyperfine tensor anisotropyis introduced.The results show that the proton superhyperfine coupling for the strongly bound axial water ligands is dominated by anisotropic interactions with the principal values of the hyperfine coupling tensor spanning a range of 18 MHz.
+
(B
1994 American Chemical Society
468
The Journal of Physical Chemistry, Vol. 98, No. 2, 1994
McCracken and Friedenberg
c
Materials and Methods
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Bis(aquo)- and bis(pyridine)tetracyanonickelate(III) complexes were prepared according to literature procedures by oxidation of Ni"(CN)42- using a 10-fold excess of hypochlorous acid.9JO Samplesused for ESEEM experimentswere mixed with 50% ethylene glycol after addition of HOC1 and frozen immediately in 4-mm-0.d. quartz EPR tubes. Typical Ni(II1) concentrations for the samples studied were 5 mM, as judged by CW-EPR. Continuous wave EPR spectra were obtained on a Varian E-4 spectrometer operatingat X-band. An EIP Model 25B frequency counter and a Micro-Now Model 5 15B NMR gaussmeter were used tocalibrate microwave frequency and magnetic field strength, respectively. Pulsed EPR studies were performed on a homebuilt spectrometerthat has been described in detail e1se~here.l~ Measurements were done at X-band using a reflectioil cavity that employed a folded stripline resonant element and a Gordon couplingarrangementsuitable for studies in a cryogenic immersion dewar.16J7 A two-pulse (goo-7-1 goo),microwave pulse sequence was used to generatethe ESEEM data used in this study. ESEEM spectra were obtained by Fourier transformation using a dead time reconstruction techniquesimilarto that described by Mimsl8 except that peak correction factors (see ref 18, eq 13) were calculated directly from the transformed spectra and entered into an interactive windowing procedure. Computer simulations of ESEEM and ENDOR spectra were performed on a Sun workstation using software written with MATLAB v4.0a (The Mathworks, Inc.).
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t a u (ueecl
RHultS
Cw-EPR spectra of Ni111(CN)4(H20)2-and Ni111(CN)4(py)~were identical to those reported by Wang and co-workers."J Both species are characterized by axial g-tensors with principal values of 2.198 and 2.007 for the bis(aquo) species and 2.14 and 2.01 for the bis(pyridine) derivative. For the bistpyridine) complex, strong superhyperfinecoupling between the axially coordinated pyridine nitrogens and the Ni(II1) ion gave rise to a five-line pattern that showed peak intensity ratios of 1:2:3:2:1. These splittings wereobserved for bothql and gl featuresof the spectrum with coupling values in good agreement with those measured previously.10 Parts a and b of Figure 1 show two-pulse ESEEM data and the corresponding ESEEM spectrum collected at g = 2.17 for Ni1I1(CN)4(H2O2)-. Sharp, low-frequency peaks are observed (Figure 1b) near 2.5,3.0, and 4.0 MHzand are assigned to cyanide I4N. The sharp positive peak at 12.5 MHz and the negative narrow line at 25.0 MHz occur at the first and second harmonic of the proton Larmor frequency at 2940 G, the field strength for which these data were collected. These two components can be assigned to matrix protons of water molecules that are close enough to the Ni(II1) center to be magneticallycoupled. The prominent negative feature at 26.1 MHz is a 'sum combination peak"I9 that is shifted from twice the proton Larmor frequency by 1.1 MHz. This peak arises from a population of protons characterized by a large, anisotropic hyperfine coupling to the nickel ion and is assigned to the axially bound water molecules where strong Ni-H dipolar couplings are expected.11-13 To help confirm the assignment of this higher frequency combination peak, parallel ESEEM studies on N~III(CN)~(PY)~were undertaken. Figure I Cshows a two-pulseESEEM spectrum taken for this derivative at g = 2.1 1. Because these data were collected at a higher magnetic field strength than used for Figure 1b, the peaks due to matrix protons have shifted to 13.5 and 27.0 MHz. The amplitudes of these frequency components are greatly reduced, indicating that the axial pyridine groups exclude much of theunbound water from thevicinityof the metalcenter. Similar to Figure lb, the spectrum of the bis(pyridine) compound is dominated by a low-frequency peak at 2.8 MHz that is assigned
I I
6
12
18
frequency
24
30
(MHz)
Figure 1. Two-pulse ESEEM data (a) and associated spectrum (b) for Ni1I1(CN)AH~0)~-colle-ctedatg= 2.17. Conditionsforthemeasurement shown in a were as follows: microwave frequency, 8.935 GHz; magnetic field strength, 2940 G; microwave pulse power, 80 W; pulse width, 16 ns (FWHM); sample temperature,4.2 K,pulse sequence repetition rate, 7 Hz;events averaged/pt, 12. Part c shows a twepulse ESEEM spectrum obtained for NiIII(CN)4(py)z- at g = 2.11. Conditiods for this measurement that differed from those of a were the following: microwave frequency, 9.250 GHz; magnetic field strength, 3135 G.
to the cyanide nitrogens. The minor peaks symmetrically displaced f2.8 MHz from the matrix proton lines at 13.5 and 27.0 MHz are combination peaks that arise from the coupling of multiple nuclei, here 14N and lH, to NilIII) as described by the 'product rule".2OJl Absent from the ESEEM data collectd for NP(CN)4(py)2- is the broad, shifted proton sum combination peak in agreement with its assignment to b u n d water for the bistaquo) complex. Two pulse ESEEM spectra collected for the bis(aquo)complex at magnetic field positions across the EPR absorption spectrum revealed shifts in the proton sum combination peak frequency that were0.5 to 1.5MHzgreater than twicetheLarmorfrequency.
The Journal of Physical Chemistry, Vol. 98, No. 2, I994 469
ESE Studies of Ni(II1)-H2O Hyperfine Interactions T
nuclear distance; h, Planck's constant; and BO,the magnetic field strength for the measurement. When the electron-proton distance is large (>3.6 A), T i s small and the sum combination peak in the two-pulse B E E M spectrum, v, YB, is resolved at 2vl. When Tis large, as it is for water protons bound axially to N P , Reijerse and Dikanov have shown for spin systems characterized by isotropic g-tensors that the v, vg peak is shifted to a frequency 16u1).12 higher than 2v1 by approximately 9P/( For orientation-selective ESEEM studies, the expression for the hyperfine fundamental frequencies of an S = l/2, I = system becomes
r
+
1.4-
+
1.2-
v(m,) = [(m,A, - vtl,)2+ (m,A, - ~
3150
3xK1
3250
3300
3350
3400
3450
3500
field strength (G) Figure 2. Plot of the shift in the proton sum combination line from twice the proton Larmor frequency versus magnetic field strength for NiI*'(CN),(H20)-. Shiftswere measured directly from the peak maxima of two-pulse ESEEM spectra collected under the following conditions: microwave frequency, 9.80 GHz; microwave power, 40 W; sample temperature, 4.2 K,pulse sequence repetition rate, 50 Hz. Each timedomain data set represented theaverageof 3Oevents. Error bars reprcscnt the inherent uncertaintyin measuring frequenciesfrom two-pulse ESEEM spectra.
~
+
1
~
)
~
(msA3 - v113)~I 'Iz (2) where m, = or -l/2 for the a and @electronspin manifolds, respectively. The other terms in eq 2 are defined below.
I, = sin e cos 4 I, = sin e sin 4 I , = COS e A , = 3gl2I1Tnln,+ g,12(g,T(3n~- 1) + a)
+ 3g3213Tn2n31/ g e
+
+
This result is expected for paramagnetic centers where anisotropy in the electron g-tensor allows complexes with different orientations with respect to the laboratory field to be sampled at each field position within the absorption ~ p e c t r u m . ' ~A, ~field ~ profile constructed by plotting the measured frequency shifts for the sum combination peak assigned to bound waters vs magnetic field strength is shown in Figure 2.
A , = 3gl21,Tn1n3 3g;l ,Tn,n3
Analysis Analysis of the field profile of Figure 2 was achieved using the formalism developedby Hutchison and McKay for single-crystal ENDOR studies23together with numerical techniques developed for the analysis of orientation- or angle-selective ENDOR exp~riments.2~.~5 Before proceeding with a brief description of our analysis, it is instructive to examine the origin of the shift in the sum combination peak for isotropic systems. For an S = l/2, I = 1/2 system where the nuclear hyperfine coupling is axial and cast in the form given by the point dipoldipole model, the fundamental hyperfine frequencies for the two electron spin manifolds are given by
11, 12, l3 are direction cosines that define the orientation of the laboratory field, BO,in the principal axis system of the g-tensor. nl, n2, and n3 define the direction of the principal axis of the hyperfine tensor with respect to the g-tensor. gl, g2, and g3 are the principal values of the electron g-tensor with g, = [g,21,2+ g22122+ g3 2 1321 1/2
= [ ( A / 2- ')IV
+ (B/2)2]1/2
uB = [(-A/2 - vI)'
+ (B/2)2]1/2
Y,
and
where
+
A = a T(3 cos' 0 - 1) B = 3 T c o s B sine and gnBnB,/h (1) The terms in the above equations are the following: VI, the Larmor frequency of the coupled nucleus; a, the Fermi contact or scalar hyperfine coupling; 8, the angle describing the direction of the laboratory magnetic field with respect to the principal axis of the hyperfine tensor; ge, the electron g-value; g,, the nuclear g-value; @,,theBohr magneton; the nuclear magneton; r, the electron*I
on,
g,l,(g,T(3n,2 - 1) + n, = sin 8, cos 4, n, = sin 6, sin 4,
n3 = cos 6,
being the effective electron g-value for a given measurement. At a fixed value of BO, the frequency position of the intensity maximum for the sum combination peak was determined by numerical simulation of the lineshape expected for this feature in a two-pulse ESEEM experiment. This task was accomplished by building up a frequency histogram for the peak followed by convolution of the histogram with a Gaussian line shape function. Possible sum combination peak frequencieswere calculated using eq 2 integrated over all possible orientations of BOthat fall on a contour of constant g,. This line integral was performed using the procedure described by Hoffman and co-workers,2* where the integral is transformed into a definite integral over I#J by numerical evaluation of the integrating factor, [(de/dd)z sin2 O]W. The amplitudes of each sum combination frequency component were computed using the method outlined by Mims26 together with this orientation averaging scheme. Simulated magnetic field profiles were constructed by plotting theshift of the sumcombination frequencycomponent from twice the nuclear Larmor frequency versus magnetic field strength. The simulationroutine was written in the MATLAB programming language and made use of the procedure described above for calculating the position of maximum intensity for the proton sum combination peak at various magnetic field positions across the EPR absorption spectrum. The first part of the calculation uses the principal values of the g-tensor and the microwave frequency used in the measurement to calculate arrays of orientations and
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41Q The Journal of Physical Chemistry, Vol. 98, No. 2, 1994
1
b
McCracken and Friedenberg principal values of 2.198, 2.198, and 2.007 and a microwave frequency of 9.80 GHz. For all three panels of the figure, the electron-nuclear distance was 2.4 A, and the scalar hyperfine coupling, a, was set to 0 MHz. For Figure 3a, 0, = 0" so that the principal axis of the hyperfine coupling tensor is aligned with g3. The resulting field profile peaks in the center of the spectrum with the maximum sum combination frequency shift occurring at 3330 G, or go = 2.10, and goes to zero at the parallel and perpendicular extremes. Qualitatively, this pattern can be understood using the results derived for isotropic spin systems by Reijerse and Dikanov,'z where the shift in the sum combination frequency was shown to depend on the product sin B cos 8. Because 0 refers to the angle between the principal axis of the hyperfine coupling tensor and the laboratory magnetic field, a maximum is expected in Figure 3a near g = 2.1 when the laboratory field sweeps out an angle of 45" with respect to the g3 axis. The Y, up frequency shift falls to zero at the high- and low-field extreme of the spectrum when 0 is 0 and 90°, respectively. When the principal axis of the hyperfine tensor is along gl (e, = 90"). Figure 3c shows that the shift falls to a minimum at the high-field extreme of the spectrum where the laboratory field is oriented along g3, making an angle of 90" with respect to the hyperfine tensor principal axis. In contrast to Figure 3a, the sum combination line frequency shift remains high at the low-field extreme of the spectrum because the laboratory field is now allowed to vary in a plane that contains the hyperfine principal axis, causing 0 to range from 0 to 277. At intermediate values of On, more complex field profiles are obtained. Figure 3b shows a field profilecalculated using the same EPR and hyperfine coupling parameters as in Figure 3a and 3c except that 0, = 45".
+
Discussion 1.2-
.
1-
0.8-
0.4 0.6
0.2
-
I
3200
3300
3400
3500
field strength (G) FIplnr3. Simulatedmagneticfield profilm for the proton sum combination peak,usingthefollowinginputparameters: g1,2.198;n,2.198;g3,2.007; microwave frequency, 9.80 GHz; g,,,5.585 36;u, 0 MHz; r, 2.4 A; 6. OD.
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For a, 0. OD, forb, e,, = 45O, and for c, 8, 90°. Shifts were computed at 20 positions across the EPR absorption envelope with the sum combination frequency obtained by averaging over the line shape in the frequency domain.
probability factors for each magnetic field position where the sum combination frequency shift will be calculated. The second part of the routine then usca a set of spin Hamiltonian parameters (gn, a, r, ,e, and e), describing the electron-nuclear coupling together with the orientation and probability weighting arrays from part 1 to compute the field profile. These two portions of the procedure can be run separately so that many sets of hyperfine coupling parameters can be tried without recalculating the orientation averaging arrays. As expected, the sum combination frequencyshift field profiles predicted by this procedure are sensitive to the orientation of the principal axis of the hyperfine coupling tensor. Figure 3 shows a set of simulations for a system with an axial g-tensor with
Use of the proton sum combination peak to characterize strong, anisotropic proton couplings to paramagnetic centers provides information that is often difficult to obtain using conventional ENDOR or ESEEM methods that focus on detection of modulations due to the fundamental (Am1 = f l ) peaks. In ESEEM experiments, anisotropy in modulation frequencies and intensities normally precludes use of the proton fundamental modulations to characterize couplingslike those typical for water molecules bound to metal centers or the a protons of .rr-radical systems. The standard approach to circumvent this problem is to perform a 2H-exchange experiment. Unfortunately, in some instances the factor of 6.5 that is given up in resolution as a result of the exchange can severely limit the amount of information gained from the st~dy.2~128A problem with proton ENDOR studies is that the region around the proton Larmor frequency is often congested and it is difficult to extract a complete powder line shape from the data, even in instances where deuteriumexchange experiments can be done to help clarify the situation. The advantages of using the proton sum combination peak in ESEEM spectra for characterizing large anisotropic proton hyperfine couplings are that the spectral lines are narrow and occur at a point in the spectrum where there is a clean spectral window, the shifted sum combination peaks can be easily detected across the entire EPR line shape with the resulting field profiles showing good sensitivity to ligand hyperfine tensor orientation, and the anisotropy in the hyperfine coupling is measured directly through its shift from twice the proton Larmor frequency. In a previous study of vandyl complexes of apoferritin, Gerfen and co-workers13utilized orientation-selective,two-pulse ESEEM experiments to detect the presenceof a bound water ligand. These authors collected ESEEM data at parallel and perpendicular extremes of the EPR spectrum and used numerical simulations of the Y, vg proton frequencies to extract hyperfine couplings and determine that the ligand was bound cis to the VO axis. In a subsequent study of VO(H20)sZ+,Tyryshkin et al.14developed analytical expressions to describe Y, + up frequency shifts as
+
ESE Studies of Ni(III)-H20 Hyperfine Interactions
The Journal of Physical Chemistry, Vol. 98, No. 2, 1994 471
a
2.56 1.4-
4
n
21.2-
W
5 4
1-
6
H
0.8-
03fb0
3&
3250
3300
&O
340
3450
\ i'
b
3500
field strength (G) Figure 4. Comparison of the field profile for the proton sum combination peak from H20 coordinated to Ni111(CN)4(H20)2-with a computersimulated profile generated using input parameters identical to those given for Figure 3, except that 6, = 12.0'. Also, the position of the sum combination line was determined from the frequency of the maximum amplitudeof the Y,, + qcomponent commensuratewith its determination from the experimental data.
10.8
-
I
, O
measured for gll and gL extremes of the EPR spectrum. Given the symmetry of the NiI11(CN)4(H202)-complex being studied here, a similar approach based on measurements at 81and gl would also suffice to determine a dipole-dipole distance for the bound water protons and the relative orientation of the principal axisof the hyperfine tensor with respect to theg-tensor. However, in a biological system where the metal site symmetry is expected to be C I ,or multiple populations of strongly coupled protons are present, a more complete study of the orientation dependence of these shifted sum combination peaks will usually be required. Therefore, we have chosen to analyze our data using the field profile format presented above.29 A computer simulation of the field profile measured for the proton sum combination peakof Ni111(CN)4(H20)2at 9.80 GHz is shown in Figure 4 along with the experimentally measured shift values. Because this complex is axially symmetric with respect to the position of the water protons and the electron g-tensor, only one population of coupled protons needed to be considered in the analysis. The range of shift values traversed in going across the EPR line shape is a strong function of the effective dipole-dipole distance which was found to be 2.4 f 0.1 A for this complex. Figure Sa shows a comparison of calculated field profiles using r = 2.2 A (solid curve), r = 2.4 A (dashed curve), and r = 2.6 A (dot-dash pattern, lower curve). Theoverall shape of the field profiles is a sensitive function of 6,. Figure 5b shows calculated field profiles where 6, is varied f 5 O from the best fit value of 12O;both give rise to poor fits of the experimental data. These field profile simulations are also sensitive to the magnitude of the scalar coupling, but the region of solution is much greater than those found for 6, and r. Figure 6 shows a pair of simulations using the same parameters as in Figure 4 except that the scalar coupling has been increased from 0 to 4 MHz for Figure 6a and to 8 MHz for Figure 6b. Our results are consistent with a range of scalar coupling values from 0 to 6 MHz. The results of the proton sum combination line analysis for axiallybound HzO in NiIII(CN)4(H20)2-yield an effectivedipoledipole distance of 2.4 A and a scalar coupling that is most likely 1 4 MHz. These values compare well with the results of singlecrystalENDOR studiesof C ~ ( H 2 0 ) 6 where ~ + the scalar couplings measured for equatorially bound water protons were
