Geometry of Reactant Centers in the CoII ... - ACS Publications

J. Phys. Chem. B 2002, 106, 8831-8841

8831

Geometry of Reactant Centers in the CoII-Substrate Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase Determined by Using Orientation-Selection-ESEEM Spectroscopy Jeffrey M. Canfield and Kurt Warncke* Department of Physics, Emory UniVersity, Atlanta, Georgia 30322 ReceiVed: March 20, 2002; In Final Form: June 10, 2002

The distances and orientations among the C5′ methyl group of 5′-deoxyadenosine, the radical-bearing C1 carbon of the substrate radical, and the low spin (S)1/2) CoII in cob(II)alamin in the active site of coenzyme B12-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized in the CoIIsubstrate radical pair state by using two-pulse X-band electron spin-echo electron paramagnetic resonance (ESE-EPR) and electron spin-echo envelope modulation (ESEEM) spectroscopies in the disordered solid state. Our approach is based on the orientation-selection created in the EPR spectrum of the biradical by the axial electron-electron dipolar interaction. Simulation of the ESE-EPR line shape yielded CoII-radical exchange and dipole interaction terms, which were used to calculate the CoII-C1 distance of 11.1 Å and the dependence of the EPR line shape on the angle between the CoII-C1 axis and the magnetic field vector. ESEEM spectroscopy performed at four magnetic field values addressed the coupling between 2H in the C5′ methyl group and the unpaired spin on C1. Global ESEEM simulations, weighted by the orientation dependence of the EPR line shape, were performed for the four magnetic fields. The C1-H distance and orientation with respect to the CoII-C1 axis are specified for each C5′ methyl hydrogen atom. In the derived model of the active site, C5′ is located close to the CoII-C1 axis (at distances of 7.8 Å and 3.3 Å from CoII and C1, respectively) while the C1-H-C5′ angle for the strongly coupled hydrogen is 165°. The near collinearity of the cobalt-carbon bond axis, radical migration coordinate and hydrogen atom transfer coordinate suggests an economy of nuclear displacements within the active site that would minimize rate-slowing molecular reorganization during the long-range radical pair separation.

Introduction The homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin (coenzyme B12, Figure 1) to produce low spin (S ) 1/2) CoII in cobalamin and a 5′-deoxyadenosyl radical species initiates radical-mediated catalysis in the adenosylcobalamin-dependent enzymes.1-6 Hydrogen atom abstraction from the carbinol carbon (C1) of the bound aminoethanol (1, RdH) or 2-aminopropanol (1, RdCH3) substrate by the C5′methylene radical center generates the substrate radical 2, with unpaired electron spin density localized on C1,11 and the C5′methyl group in 5′-deoxyadenosine. The substrate radical SCHEME 1

rearranges to a product radical, and a second hydrogen atom transfer, from C5′ to C2 of the product radical, yields the diamagnetic product and reforms the 5′-deoxyadenosyl radical. The 5′-deoxyadenosyl radical and CoII may then recombine.12 The reaction sequence is shown schematically in Figure 2. The * Corresponding author. Department of Physics, N201 Mathematics and Science Center, 400 Dowman Drive, Emory University, Atlanta, GA 30322. Tel: 404-727-2975.Fax: 404-727-0873.E-mail: [email protected].

participants in the radical migration in B12 enzymes were originally inferred from steady-state hydrogen isotope exchange and electron paramagnetic resonance (EPR) studies.1,2,4 The purpose of the present work is to gain insight into the mechanisms of radical migration, hydrogen atom transfer, and radical rearrangement by using high-resolution techniques of pulsed-EPR spectroscopy14-17 to reveal the structure and arrangement of the reactants in the Class II adenosylcobalamindependent enzyme, ethanolamine deaminase1,18,19 (also known as ethanolamine-ammonia lyase) from Salmonella typhimurium, which catalyzes the conversion of aminoethanol to acetaldehyde and ammonia.20 The CoII-substrate radical pair state that accumulates during steady-state turnover on (S)-2-aminopropanol in ethanolamine deaminase can be cryotrapped in high yield,11 and lowtemperature EPR studies of this state in disordered samples have already yielded structural information. EPR simulations21 of the powder pattern radical pair line shape11 in ethanolamine deaminase from Clostridium sp. showed that C1 and CoII are separated by 10-12 Å along a direction approximately parallel to the CoII dz2 orbital axis. This direction corresponds to the z-component of the CoII g-tensor, which is perpendicular to the plane of the corrin ring in cob(II)alamin.22 High-resolution pulsed-EPR techniques of electron spin-echo envelope modulation (ESEEM)23 and ESE-electron-nuclear double resonance (ENDOR),24 performed on the substrate radical state, have revealed that C1 and C5′ are separated by 3.2-3.3 Å. These results provide evidence that the C5′ carbon atom of the 5′-

10.1021/jp0207634 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/06/2002

8832 J. Phys. Chem. B, Vol. 106, No. 34, 2002

Figure 1. Depiction of the structure of coenzyme B12 (adenosylcobalamin). The X-ray crystallographic structures of cobalamins have been reviewed.7,8 The β-axial ligand is the 5′-deoxyadenosyl group (above page plane) and the R-axial ligand is 5,6-dimethylbenzimidazole (below page plane). The coenzyme retains dimethylbenzimidazole as R-axial ligand when bound in ethanolamine deaminase.9,10 R1 and R2 refer to acetamide and propionamide side chains. The C5′ carbon of 5′deoxyadenosyl is labeled, and the two exchangeable hydrogen atoms on the coenzyme are shown enlarged in bold.

Figure 2. Minimal mechanism of radical migration in vitamin B12 coenzyme-dependent enzymes.3,4,13 The forward direction of reaction is indicated by arrows. Substrate-derived species in the radical pair states are designated S-H (bound substrate), S‚ (substrate radical), P‚ (product radical), and P-H (bound products). The 5′-deoxyadenosyl β-axial ligand is represented as Ad-CH2- in the intact coenzyme, and as Ad-CH2• (5′-deoxyadenosyl radical) or Ad-CH3 (5′-deoxyadenosine) following cobalt-carbon bond cleavage. The cobalt ion and its formal oxidation states are depicted, but the corrin ring and R-axial ligand of the coenzyme are not shown for clarity.

deoxyadenosyl moiety exchanges hydrogen directly with substrate and product species.23,24 ESEEM from the interaction of the unpaired electron spin on C1 with 2H nuclei incorporated specifically into the C5′ methyl group also showed that one C5′ hydrogen atom was positioned 2.2 Å from C1 and approximately along the C1-C5′ axis.23 The other two methyl hydrogen atoms were located 3.8 Å from C1. To accurately determine the displacement of C5′ from CoII in the radical pair state, the relative distances and orientations between CoII in the

Canfield and Warncke cobalamin binding region and the reactant centers (C1, C5′) in the substrate binding region must be determined. The present work addresses the three-dimensional geometry of reactant centers in the active site of ethanolamine deaminase by using the orientation-selection that is created in the EPR line shape of the substrate radical component of the biradical spectrum by the anisotropic electron-electron dipolar interaction. Different magnetic field values across the substrate radical EPR line shape correspond to restricted sets of orientations of 98 the electron-electron (R B ) CoII-C1) vector relative to the external magnetic field vector. The orientations contributing to the spectrum at any magnetic field value can be calculated from simulations of the EPR spectrum. ESEEM collected at a particular magnetic field value is representative of the corresponding set of restricted orientations. The electron-nuclear interactions that give rise to the ESEEM include an anisotropic contribution from the dipolar hyperfine interaction that is sensitive to the orientation of the electron-nuclear vector relative to the external magnetic field. This is the case for the interaction of the unpaired electron on C1 with 2H nuclei incorporated specifically into C5′, as indicated by the dipolarbroadened 2H ESEEM from these nuclei.23 Therefore, the 98 orientation of the electron-electron (CoII-C1) and electron98 nuclear (C1-2H) vectors can be determined by using the simulated EPR orientation-selection to weight the contribution of different orientations to the ESEEM. The experimental and theoretical approaches are related to orientation-selection (polycrystalline) ENDOR25-29 and ESEEM30 spectroscopy in transition metal systems, where the orientation selection is created primarily by anisotropy of the g-tensor. To our knowledge, the present study represents the first application of ESEEM to probe orientation-selection in a disordered system created by electronelectron dipolar anisotropy. We present an approach for extracting molecular structure information from the modest orientation-selection in the EPR spectrum of biradicals in the intermediate coupling regime. Global simulation analysis of the 2H-ESEEM collected at four values of the magnetic field across the substrate radical EPR line shape reveals the orientation of the three C5′ methyl hydrogen positions relative to the CoII-C1 axis. The ESE-EPR simulations give a value of 11.1 Å for the CoII-C1 separation in S. typhimurium, in agreement with the value determined for the enzyme from Clostridium sp.21 A model for the threedimensional geometry of the 2H nuclei, C1, C5′, and CoII is presented, in which C5′ and the strongly coupled hydrogen nucleus lie approximately along the CoII-C1 axis. The coordinates of radical migration and hydrogen atom transfer suggested by this model provide insights into the mechanism of radicalmediated catalysis in ethanolamine deaminase. Experimental Procedures Enzyme Preparation. Enzyme was purified from the Eschericia coli overexpression strain incorporating the cloned S. typhimurium ethanolamine deaminase coding sequences31 essentially as described,32 with the exception that the enzyme was dialyzed against buffer containing 100 mM HEPES (pH 7.45), 10 mM KCl, 5 mM dithiothreitol, 10 mM urea, and 10% glycerol.33 Enzyme activity was determined as described34 by using the coupled assay with alcohol dehydrogenase/NADH. The specific activity of the purified enzyme with aminoethanol as substrate was 35 µmol/min/mg. Sample Preparation. Adenosylcobalamin (Sigma Chemical Co.), 1,1,2,2-2H4-amino-ethanol (Cambridge Isotope Laboratories, Inc.), and (S)-2-aminopropanol and natural abundance

Reactant Center Geometry in Ethanolamine Deaminase aminoethanol (Aldrich Chemical Co.) were purchased from commercial sources. The reactions were performed in airsaturated buffer containing 100 mM HEPES (pH 7.5), 10 mM KCl, and 5 mM dithiothreitol. Identical results were obtained with air-saturated and anaerobic samples. All manipulations were carried out on ice under red safe-lighting. The substrate radical was generated by using a procedure for fast cryotrapping of steady-state intermediate states in ethanolamine deaminase.35 The final concentration of enzyme was 30 mg/ml, which is equivalent to 60 µM for a holoenzyme molecular mass of 500 000 g/mol.32 Adenosylcobalamin was added to 360 µM, which is stoichiometric with active sites. The active site/ holoenzyme stoichiometry of 6 is based on adenosylcobalamin titrations of substrate radical formation (K. Warncke, unpublished), and is in agreement with the value obtained by two separate methods.36,37 The procedure for specific incorporation of 2H into the hydrogen positions of the C5′ carbon in enzymebound adenosylcobalamin has been described in detail.23 Briefly, in a 2H-prelabeling reaction with 2H4-aminoethanol, and parallel control reaction with natural abundance aminoethanol, turnover was initiated by addition of adenosylcobalamin to premixed enzyme and 36 mM aminoethanol, and conversion of aminoethanol to products was allowed to proceed to completion (reaction for 2 min). This pretreatment leads to exchange of 2H into C5′ of the coenzyme.23 (S)-2-aminopropanol (10 mM) was then added to the sample, and after an additional 12-15 s time interval, during which the sample was loaded into a 4 mm o.d. EPR tube, the sample was plunged into liquid nitrogen-chilled isopentane (T = 130 K) to trap the substrate radical state. ESE-EPR Data Acquisition and Processing. ESE-EPR spectra were collected by using a home-constructed wideband pulsed-EPR spectrometer that will be described elsewhere (K. Warncke, in preparation). The reflection microwave probe38 features a folded half-wave microwave resonator.39 ESE-EPR spectra were obtained by using the two-pulse microwave pulse sequence.14-17 All data processing and analysis was performed with routines written in Matlab (version 6.0, Mathworks, Natick, MA) and run on PowerMacintosh or PC computers. ESEEM Data Acquisition and Processing. ESEEM was collected by using the two-pulse microwave pulse sequence.14-17 Envelope modulation was deadtime-reconstructed40 and cosine Fourier transformed to generate ESEEM frequency spectra. All data processing and analysis was performed with routines written in Matlab.

J. Phys. Chem. B, Vol. 106, No. 34, 2002 8833

Figure 3. Coordinate systems used to define the relative positions of the CoII, C1, C5′ and C5′ methyl group hydrogen atoms in the active site of ethanolamine deaminase. (A) The xyz coordinate system used to define the orientation of the magnetic field vector (Bo) relative to the active site in ESE-EPR and ESEEM simulations. (B) Relationship of the xyz coordinate systems used to describe the positions of the atom centers in the active site. (C) The orientation of the C5′ methyl group hydrogens in the x1y1z1 coordinate system from (B).

C1, CoII, and the three hydrogen atoms. The unit vectors, xˆ j, yˆ j, and zˆj, for j ) 1 and 2 are parallel, θ1 and φ1 are the polar and azimuthal angles for the displacement vector from C5′ to C1 in the x1y1z1 coordinate system, and θ2 and φ2 are the polar and azimuthal angles in spherical coordinates for the displacement vector from C1 to CoII in the x2y2z2 coordinate system. CoII is a distance R from C1. Figure 3C shows the same x1y1z1 coordinate system as in Figure 3B, but with the three C5′ methyl group hydrogen atoms. Since C5′ is assumed to be sp3-hybridized, each hydrogen atom Hn is a fixed distance of 1.1 Å from C5′, and the Hn-C5′-Hm angle is fixed at 109.5° for n * m. To obtain the βn and rn values needed for ESEEM calculations for a particular set of (θ1,φ1) and (θ2,φ2), the atom positions are calculated and the following expressions are used:

98 rn ) | C1-Hn| -cos(βn) )

(1)

9 8 98 9 8 98 CoII-C1‚ C1-Hn CoII-C1‚ C1-Hn (2) ) 9 8 98 R‚rn II | Co -C1|| C1-Hn|

Theory Coordinate Systems. Figure 3 shows the three coordinate systems that specify the orientations and separation distances among the atom centers. Figure 3A shows the xyz coordinate system used for ESE-EPR and ESEEM simulations. Here, θ and φ are the polar and azimuthal angles in spherical coordinates that describe the orientation of the magnetic field vector B B0. Note that CoII is at the origin, while C1 lies along the z axis a distance R from CoII. The three C5′ methyl hydrogen sites are denoted as Hn, where n ) 1-3. H1 refers to the strongly coupled, and H2,H3 to weakly coupled, hydrogen sites. Each Hn is treated as if it lies in the xz plane a distance rn from C1 because the EPR spin Hamiltonian (eq 17) is axial about zˆ and all orientations θ, φ of B B0 are sampled in the powder average. The Hn-C1-CoII angle for each hydrogen is denoted βn. By symmetry, βn and β′n ) 180° - βn give the same ESEEM waveform. Thus, only βn from 0 to 90 ° need be considered. Figure 3B shows the Cartesian coordinate systems x1y1z1 and x2y2z2 that are used to calculate the relative positions of C5′,

Since θ1 and r1 have a one-to-one relationship, r1 can be fixed to a particular value by fixing θ1 as below:

cosθ1 )

98 98 98 | C5′-H1|2 + |C1-C5′|2 - |C1-H1|2 98 98 2| C5′-H1|| C1-C5′|

(3)

Powder Averaging and Angle Selection. Simulations of ESE-EPR and ESEEM spectra from frozen solutions such as those used in this study can be obtained by using powderaveraging. If f (θ,φ) is the EPR spectrum that a single crystal would give if the steady field B B0 were oriented in the θ,φ direction with respect to the crystal’s xyz axes, as shown in Figure 3A, then the powder average is given by

∫0πsin(θ) dθ∫02πdφ f(θ,φ) 〈f (θ,φ)〉 ) ∫0πsin(θ) dθ∫02πdφ

(4)

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Canfield and Warncke

In practice, this average must be approximated numerically by summing over a finite set of orientations θj,φj, where each orientation θj,φj represents a range of orientations from θj,min to θj,max and φj,min to φj,max that includes θj,φj. This gives

∑j ∫θ

θj,max

sin(θ) dθ

j,min

〈f (θ,φ)〉 =

∑j ∫θ

θj,max

yj ) Axj + B + Cj

j,max

j,min

sin(θ) dθ

j,min

)

∫θθ

points Yj) and a calculated curve (the points xj). The three fitting functions σ, r, and  that are used are described below. The calculated curve is given by the following general expression

dφ f(θj,φj)

∫θ

(5)

θj,max

dφ

j,min

∑j [cos(θj,min) - cos(θj,max)](φj,max - φj,min)f(θj,φj) ∑j [cos(θj,min) - cos(θj,max)](φj,max - φj,min)

The coefficient, A, scales the range of xj, B accounts for a constant “baseline” contribution, and Cj accounts for a linear drift in the baseline. The fitting function, σ, is given by the squared deviation between Yj and yj, as follows:

σ) (6)

〈f(θ,φ)〉 =

(7)

∑j sin(θj) dθj dφj

∫0πsin(θ) dθ∫02πdφ F(θ,φ) 〈F(θ,φ)〉 ) ∫0πsin(θ) dθ∫02πdφ

(8)

∫0 sin(θ) dθ∫0 dφ F(θ,φ)P(θ,φ) 〈F(θ,φ)〉 ) ∫0πsin(θ) dθ∫02πdφ P(θ,φ)

(9)

〈F(θ,φ)〉 = [cos(θj,min) - cos(θj,max)](φj,max - φj,min)F(θj,φj)P(θj,φj)

∑j

∑j [cos(θj,min) - cos(θj,max)](φj,max - φj,min)P(θj,φj) (10)

∑j sin(θj) dθj dφj F(θj,φj)P(θj,φj) ∑j sin(θj) dθj dφj P(θj,φj)

(11)

Fitting Procedures. To fit the experimental EPR and ESEEM data, the Nelder-Mead simplex direct search method,41,42 as implemented in Matlab’s “fmins/fminsearch” algorithm, was used. This algorithm minimizes a fitting function that reflects the goodness of fit between an experimental curve (the data

]

∑j - 2AxjYj - 2BYj - 2CYj j

+ 2ABxj + 2ACxj j + 2BCj

∑(2Axj2 - 2xjYj + 2Bxj + 2Cxj j) ) 0, j

where

d2σ dA

dσ

)

∑(2xj2) g 0 j

j

d2σ

where

dσ

)

2

∑(2B - 2Yj + 2Axj + 2Cj) ) 0,

)

dB2

)

∑(2) g 0 j

∑(2Cj2 - 2Yj j + 2Axj j + 2Bj) ) 0, j

where

d2σ 2

dC

Furthermore, the orientation-selected ESEEM analogues of eqs 6 and 7 are as follows:

〈F(θ,φ)〉 =

)

dA

dC

2π

[

(13)

which at its minimum gives

dB

To account for the orientation selection of the ESEEM, the factor P(θ,φ), which reflects the relative population, or relative EPR transition intensity, at each orientation θ,φ, enters eq 8 as follows: π

)

dσ

Similarly, if F(θ,φ) is the ESEEM waveform that a single crystal would give, the powder-averaged ESEEM waveform is given by an expression comparable to eq 4, as follows:

∑j (Yj - yj)2 ) ∑j [Yj - (Axj + B + Cj)]2 Yj2 + A2xj2 + B2 + C2j2

which, if dθj ) θj,max - θj,min and dφj ) φj,max - φj,min are sufficiently small, gives

∑j sin(θj) dθj dφj f(θj,φj)

(12)

)

∑(2j2) g 0

(14)

j

The first derivative expressions in eq 14 can be solved to find the set of A, B, and C values that gives the smallest σ value for a particular set of experimental Yj and calculated xj. Using eq 12 with C ) 0 matches the shape of an experimental spectrum, but neglects any base-line drift. This function was used to obtain σ values when fitting the ESE-EPR spectra. A fitting function with a nonzero C value was used in fitting the ESEEM waveforms. In this case, the rationale for the Cj form of the drift term is accounted for as follows. The experimental waveform data, Yj, where j ) τ, are the ratio of two approximately exponential43 decays. If the decay rates k1 and k2 are slightly different, as is approximately true for the quotient ESEEM waveforms examined here, the following would hold:

Yj ) e-k1j/e-k2j ) e-(k1 - k2)j = 1 - (k1- k2)j

(15)

The fitting of the ESEEM waveforms was based on the fitting function r, or Pearson’s correlation coefficient, also known as the product-moment or linear correlation coefficient.41,44,45 The value of r is defined as

Reactant Center Geometry in Ethanolamine Deaminase

r)

J. Phys. Chem. B, Vol. 106, No. 34, 2002 8835

∑j (xj - xj)(Yj - Yh )

x

(16)

x

∑j (xj - xj)2 ∑j (Yj - Yh )2

where xj is the mean of the xj and Y h is the mean of the Yj. Pearson’s r can be used to match a spectrum’s shape while neglecting constant offsets. The value of r is +1 for highly correlated data and 0 for uncorrelated data. A fitting function,  ) 1 - r2, was applied to the global fitting of the ESEEM waveforms at different magnetic fields. The value of  approaches one for poor fits and drops to zero for the best fits. This function has the advantage over r of having greatest resolution near the best-fit parameter set. Global (simultaneous) fitting of ESEEM waveforms collected at different Bo values was performed by minimizing the sum of  values from the waveforms for each field Bo. ESE-EPR Simulations. The program MENO46,47 and the frequency-based “direct-diagonalization” method in the program DSNANI48 were used to simulate the ESE-EPR spectra. Both programs treat spin Hamiltonians such as those previously reported for radical pairs in four different coenzyme B12dependent enzymes21 that contain axial dipolar interaction terms, isotropic J exchange terms, and axial g and A hyperfine tensors. The Hamiltonian is written as

B2 + A⊥(S1xI1x + S1yI1y) + A| S1zI1z] + H ) h[-JS B1·S µ 2)‚B Bo + (µ b1 + b

[

]

µo b b1‚R µ 1‚µ b2 3(µ B)(µ b2‚R B) (17) 4π R3 R5

where

b µ j ) βe(gjxSjxxˆ + gjySjyyˆ + gjzSjzzˆ)

(18)

The subscripts “1” and “2” correspond to CoII and the radical, respectively. The value of J has magnitude 240-420 MHz depending on the enzyme,21 B R is along zˆ, g1x ) g1y ) g⊥ ) 2.27, g1z ) g| ) 1.99, g2xyz ) 2, S1 ) S2 ) 1/2, and I1 ) 7/2. The cobalt hyperfine parameters are A⊥ ) 30 MHz (10 × 10-4 cm-1) and A| ) 309 MHz (103 × 10-4 cm-1).21 The sign convention for J is identical to that used in MENO,46 but opposite to that used in DSNANI.48 In MENO, negative J indicates antiferromagnetic coupling, and thus corresponds to a lower energy for the singlet relative to the triplet state.46 Other hyperfine interactions were neglected. For the radical, this was in part because there is no resolved hyperfine splitting in the radical line shape. Isotope substitutions on the substrate radical have shown effects on the radical line width (20 mT) that are small relative to J and the dipole interaction D.11,49 For example, substitution of 2H for 1H at the R-hydrogen position on C1 results in a line width reduction of 1.0 mT.11 In addition, hydrogen isotope substitutions on C5′ do not perturb the line shape.23 As described in Results and Discussion, we have performed simulations that explicitly incorporate the strong R-1H hyperfine interaction. The results show that the inclusion of specific, strong hyperfine couplings does not significantly influence our conclusions. In both MENO and DSNANI, different Gaussian line widths were used for the CoII and radical contributions to the spectrum to account for the different relaxation behavior and unresolved hyperfine couplings for each electron.11 The intensity in the CoII region relative to the radical intensity is attenuated in ESEEPR, owing to the more rapid relaxation (shorter Tm) of CoII relative to the organic radical. The loss of CoII intensity in the

ESE-EPR spectrum was exacerbated by the relatively long value of τ used. The τ value of 400 ns was chosen because it gives maximal ESE amplitude for the 2H-labeled sample. These features of the ESE experiment are not accounted for by the simulation, even when the line width parameters for CoII and the radical are varied separately. Therefore, to obtain the best possible fit, the CoII contributions were further reduced by using an additional scaling factor. For powder-averaging, DSNANI used eq 7, while MENO used eq 6. Finally, since MENO originally output first derivative spectra, its code was adapted to analytically integrate the line shape function (roughly as in eq K.45, p 194 of ref 48). The results were tested by comparing with DSNANI results and numerically integrated MENO results. To optimize the fit, J and R were varied from a variety of initial parameter sets to minimize σ as specified in eq 13, but with C ) 0 and xj the direct output from MENO. Although both J and R contribute to the magnitude of the doublet splitting in the radical line shape, R also determines the anisotropic part of the dipole interaction, and through this R also influences the line width of each Pake doublet feature. Comparisons of MENO and DSNANI results showed agreement in σ values and spectral appearance. Since DSNANI’s frequency-based “direct diagonalization” method obtains the true eigenvalues and eigenvectors at each field position, the agreement between MENO and DSNANI simulations shows that the perturbation series used to derive MENO holds quite well for spin Hamiltonians such as the one used here. EPR absorption spectra for single orientations (as shown in Figure 5) were calculated by using MENO based on the bestfit powder pattern EPR spectrum. A series of EPR absorption spectra was calculated for each θ value, and cross-sections of the EPR absorption versus orientation at the particular magnetic field values of 306.5, 310.5, 316.5, and 322.0 mT were taken. The cross section at each field was rescaled to have a maximum of 1, and each resulting cross section (as shown in Figure 6) was denoted as P(θ,φ). Since the area under an EPR absorption spectrum can be used to measure the spin population,50 the resultant P(θ,φ) curves were then used to weight the powder average, as given by eq 10, when calculating the ESEEM spectra. Using P(θ,φ) proportional to the EPR absorption (f (θ,φ) in eqs 4-7) when powder averaging the ESEEM gives equations very much like eqs 1 and 3 in ref 51, where an ENDOR spectrum contains a factor due to the EPR absorption spectrum. ESEEM Simulations. IndiVidual Electron-Nuclear Couplings. The ESEEM simulation approach was based on Mims’ formalism,52-54 and the implementation of the approach has been described.23 Briefly, the simulation focused on the coupling of the unpaired electron spin on C1 with the three fixed hydrogen sites on the C5′ methyl group. Each hydrogen site was parametrized by rn, βn, and Aiso,n, the isotropic hyperfine coupling with C1. The ESEEM owing to each hydrogen site was then calculated separately by using the spin Hamiltonian w

H ) βe geB Bo·S B2 - βNgNB Bo·BI 2 + hS B2· A2·BI 2

(19)

where ge ) 2, S2 ) 1/2, gN ) 5.5857 for 1H or 0.8574 for 2H, w I2 ) 1/2 for 1H or 1 for 2H, and A2 is the complete hyperfine tensor. The nuclear quadrupole interaction terms for the I ) 1 2H nucleus were neglected because they are generally small compared to the hyperfine couplings and had little influence w on simulations in preliminary work. Note that A2 is rotated 9 8 thru the Euler angle βn into the basis where CoII-C1 is along w the z axis from a basis where A2 has the diagonal elements

8836 J. Phys. Chem. B, Vol. 106, No. 34, 2002

Canfield and Warncke

gN + [-Adip,n, - Adip,n, 2Adip,n] A 0.8574 iso,n

(20)

where

Adip,n )

µo βe ge βNgN 1 g e gN ) (7.069 MHz Å3) 3 (21) 3 4π h r r n

n

w

Once each site’s hyperfine tensor A2 was rotated by βn to the 98 CoII-C1 basis, it was straightforward to weight each orientation’s ESEEM as in eq 10 by using P(θ), the θ-dependence of the ESE-EPR simulation. This allowed the treatment of effects on the ESEEM due to J, R, and CoII without explicitly including such terms in the spin Hamiltonian. Combination of Electron-Nuclear Couplings. The overall two-pulse ESEEM for a group of nuclei interacting with the same electron is given by the product of the ESEEM obtained from each nucleus separately.52,53 This product rule treats each nucleus independently and allows envelope division of the ESEEM waveforms from two different samples to reveal the effects of just those nuclei that differ between the two samples. For disordered samples in the case of weak hyperfine interactions, powder-averaging the ESEEM from each nucleus is performed separately and then the individual powder-averaged ESEEM's are multiplied to obtain the overall total ESEEM.55,56 The ESEEM from each coupled C5′ hydrogen site was powder-averaged or angle-selected. The resultant waveforms were denoted E H1 - E H3 for protium-occupied sites and E D1 E D3 for deuterium-occupied sites. Following our previous method23 to account for the three possible permutations of two 2H and one 1H among the three hydrogen sites on C5′, these signals were multiplied into three different products, E D1 E D2 E H3 , E D1 E H2 E D3 , and E H1 E D2 E D3 . Triply deuterated methyl groups were not considered, because one hydrogen on the C5′ methyl group is expected to come from the natural abundance 2-aminopropanol. The overall ESEEM waveform from the 2H-labeled sample is

1 ETOT ) (E D1 E D2 E H3 + E D1 E H2 E D3 + E H1 E D2 E D3 ) 3

(22)

Envelope-dividing eq 22 by the ESEEM from the sample prepared by using natural abundance, approximately all-1H, aminoethanol gives the overall ESEEM quotient waveform

1 QTOT ) (Q1Q2 + Q1Q3 + Q2Q3) 3

(23)

where Q1 ) E D1 /E H1 , Q2 ) E D2 /E H2 , and Q3 ) E D3 /E H3 . When fitting the ESEEM waveforms, τ values from 180 to 1674 ns were used to calculate residuals, σ, and correlation coefficients, r. This range of τ values reduced effects from (a) deadtime artifacts for initial time points and (b) envelope decayinduced drops in signal-to-noise for late time points. To obtain FT spectra from both experimental and calculated data, τ values from 180 to 2418 ns from envelope-divided spectra were used, and the deadtime corresponding to 0-180 ns was reconstructed.40 To compare FT’s from experimental and calculated spectra, residuals σ and correlation coefficients r were calculated using frequency points from 0.5 to 9.9 MHz. This focused on the frequency regions that include the 2H features. Results and Discussion ESE-EPR Spectroscopy. Figure 4 shows the experimental ESE-detected EPR absorption spectrum of the CoII-substrate

Figure 4. X-band two-pulse ESE-EPR spectrum of the CoII-substrate radical intermediate cryotrapped in ethanolamine deaminase. The enzyme was pretreated with natural abundance aminoethanol, and the substrate radical was formed from natural abundance (S)-2-aminopropanol. The line shapes for 2H- and 1H-prelabeled enzyme are the same.23 The free electron resonance position at g ) 2.0 is shown by the arrow. Experimental conditions (solid lines): τ, 400 ns; microwave frequency, 8.968 GHz; temperature, 6 K; microwave pulse power, 10 W; microwave pulse width, 100 ns; pulse sequence repetition rate, 20 Hz; 32 repetitions averaged per point; average of four individual spectra. Simulation parameters (dotted lines): J, -324 MHz; R, 11.1 Å; line widths, 4.87 mT (radical), 13.9 mT (CoII); CoII scale factor, 0.27.

TABLE 1: Best-Fit ESE-EPR Simulation Parameters and Fit Numbers Obtained by Using MENO for Figure 4a input parameters

best-fit values

ranges around best fits

J/MHz R/Å radical line width/mT CoII line width/mT CoII scale factor

-324 11.1 4.87 13.9 0.27

-325--322 10.9-11.5 4.80-4.92 11.0-16.6 0.25-0.28

fitting parameters

best-fit values

ranges around best fits

r σ/10-15

0.996 5.81

0.996-0.996 5.81-5.85

a The minimum and maximum values for each parameter represent the range obtained when the indicated parameter was varied with all the other parameters held fixed at their best-fit values. These extreme values reflect the parameter range (to the number of digits shown) in which r remains fixed (when rounded to three significant digits) at its best-fit value. For r and σ, the ranges represent values obtained within the input parameter ranges.

radical pair state. The region of greatest CoII intensity is at low magnetic field, from approximately 270-300 mT, corresponding to the g⊥ ) 2.27 position of the CoII transitions in free cob(II)alamin.22 EPR transition intensity associated with the substrate radical appears at 310-335 mT, around g ) 2.0. The partially resolved doublet splitting of the radical line shape arises from the coupling of the unpaired electron spins localized on CoII and C1. The doublet splitting generally increases as |J| rises, and an examination of the dependence of biradical line shapes on J has been reported.57 For J ) 0 and R g 9 Å, the doublet merges into a central peak. The agreement between the experimental and simulated line shapes in Figure 4 is very good. The best-fit parameters and ranges giving comparable r values are listed in Table 1. The fit shows two large best-fit basins of attraction, one for J ) 324 MHz with R ) 11.1 Å and the other for J ) 335 MHz

Reactant Center Geometry in Ethanolamine Deaminase

J. Phys. Chem. B, Vol. 106, No. 34, 2002 8837

Figure 5. Powder and single-orientation ESE-EPR simulations for the substrate radical intermediate in ethanolamine deaminase. The four vertical dotted lines mark the magnetic field values at which ESEEM was collected. The dotted-line spectra represent P(θ) and the solid lines correspond to P(θ) weighted by sin θ. The free electron resonance position at g ) 2.0 is shown by the arrow. Simulation parameters: J, -324 MHz; R, 11.1 Å; line widths, 4.87 mT (radical), 13.9 mT (CoII); CoII scale factor, 0.27.

with R ) 115 Å. To relate J and R for radical pairs, Coffman and Buettner58 derived the following empirical expression

|J| e 4.05 × 1011e-1.8R

(24)

where |J| is in MHz and R is in Å. The value of J ) -324 MHz yields R e 11.6 Å, which is consistent with the R ) 11.1 Å obtained here. The best-fit J and R values are comparable to the values determined previously by Boas et al.21 for the CoIIsubstrate radical pair in ethanolamine deaminase from Clostridium (R ) 10-12 Å; antiferromagnetic J of magnitude 240 MHz, or J ) -240 MHz in the convention used here). The larger magnitude of J for the CoII-substrate radical interaction in our study reflects the different temperature of spectrum acquisition, which was 6 K for the ESE-EPR and 90 K for the CW-EPR studies,11 upon which the earlier simulations21 were based. Previous studies have shown that the radical doublet splitting increases as the temperature decreases in ethanolamine deaminase.49 This effect was also observed in B12-dependent ribonucleotide reductase.59 Quite different parameters of J ) -12.6 to -18.2 MHz (in the convention used here) and R ) 13 Å were obtained for the interaction of CoII and a hydrazine cation radical fragment bound near the substrate binding site.37 Anisotropy of the EPR Line Shape: Field Dependence of the Spectra at Different Orientations. The calculated spectral contributions of different single orientations θ of the electronelectron vector B R relative to B Bo are shown in Figure 5. These single orientation spectra were calculated by using the parameters obtained in the fit shown in Figure 4. In Figure 5, the upper left panel shows the calculated ESE-EPR absorption spectrum that is powder-averaged over all orientations, as in Figure 4. The other panels show curves for ESE-EPR absorpton spectra calculated at individual θ orientations (dotted lines). The solid curves show the result of multiplying the dotted curves by the sin θ weighting factor from the powder-average in eq 4. Thus, the solid curves show which orientations contribute most to the powder average. For example, the orientations near B R perpendicular to B Bo are most probable and therefore the powderaverage spectrum looks more like the single orientation spectrum calculated at 90° than that at 1°. In Figure 5, the features arise

Figure 6. Relative contributions of orientations, θ, to the EPR transition intensity of the substrate radical intermediate in ethanolamine deaminase at the four values of fixed magnetic field used for ESEEM. The dottedline spectra represent P(θ), and the solid lines correspond to P(θ) weighted by sin θ. Simulation parameters: J, -324 MHz; R, 11.1 Å; line widths, 4.87 mT (radical), 13.9 mT (CoII); CoII scale factor, 0.27.

predominantly from the organic radical. This was shown by varying the simulation line widths, by the negligible effect on the radical line shape of dropping the CoII scaling factor to zero, and by direct examination of the origin of transition intensities in MENO. Anisotropy of the EPR Line Shape: Orientation Dependence of the Spectra at Different Magnetic Fields. Figure 6 shows the contribution of different orientations θ to the ESEEPR spectrum at the four magnetic field positions marked by vertical dotted lines in Figure 5. The ESEEM experiments, discussed below, were performed at these magnetic field values. In Figure 6, the curve at each magnetic field is normalized so that the maximum is 1, and the result is denoted P(θ). The dotted curves represent the raw P(θ) value, while solid curves include the sin θ weighting factor, so that they reflect the overall contribution that each orientation makes to the powder average. The sin θ factors serve to amplify the dominance of θ orientations near 90° for 306.5, 310.5, and 322.0 mT. The sin θ factor also suppresses contributions from θ < 45°. Thus, at 316.5 mT, while the P(θ) curve includes significant contributions from orientations below 30°, the P(θ) sin θ curve has a maximum near 45°. The low-amplitude features at θ ) 22-23°, 34-35°, and 4243° in Figure 6 are artifacts of the MENO perturbation series method, and are caused by the reassignment of transitions between CoII and radical and the different line widths used for CoII and radical. A detailed description of this effect and a table of the transitions involved are given in Supporting Information (Table S1 and legend). The P(θ)sinθ curves in Figure 6 reflect the relative spin population at each orientation and so can be used as in eq 10 to weight the ESEEM powder averages. The EPR simulation results presented in Figures 5 and 6 show that the low field edge of the radical EPR line shape is dominated by perpendicular orientations of the CoII-C1 vector relative to B Bo. Therefore, the hyperfine line shapes in an ESEEM experiment performed at 306.5 mT on the low field edge will be representative of these orientations. The central region of the radical EPR line shape around 316.5 mT corresponds to the high field edge of the larger amplitude low-field Pake doublet feature, and therefore, the intensity here is dominated by more nearly parallel

8838 J. Phys. Chem. B, Vol. 106, No. 34, 2002

Figure 7. Experimental two-pulse 2H/1H quotient ESEEM collected from the (S)-2-aminopropanol radical in ethanolamine deaminase at four different magnetic field values (solid lines), and corresponding overlaid simulations (dotted). The dark horizontal bars show the range of data that were used in the fitting procedure. (A) Bo ) 306.5 mT. (B) Bo ) 310.5 mT. (C) Bo ) 316.5 mT. (D) Bo ) 320.0 mT. Experimental conditions: temperature, 6 K; microwave frequency, 8.83 GHz; microwave pulse power, 40 W; initial τ value, 180 ns; τ increment, 6 ns; π/2 pulse width, 20 ns; pulse repetition rate, 64 Hz; 100 repetitions averaged per point; average of six envelopes. Simulation fitting parameters: All as defined in eqs 12 and 16: (A) r ) 0.9624, A ) 0.77, B ) 0.58, C ) -0.095 µs-1, -1/C ) 11 µs. (B) r ) 0.9869, A ) 0.94, B ) 0.45, C ) -0.058 µs-1, -1/C ) 17 µs. (C) r ) 0.9104, A ) 0.58, B ) 0.63, C ) -0.0024 µs-1, -1/C ) 420 µs. (D) r ) 0.9602, A ) 0.95, B ) 0.50, C ) -0.081 µs-1, -1/C ) 12 µs.

orientations of the CoII-C1 vector relative to B Bo. Between these magnetic field values at 310.5 mT, the full range of CoII-C1 versus B Bo orientations are sampled, and the ESEEM spectrum will correspond closely to a powder pattern. The same is true for the magnetic field value of 322.0 mT, which corresponds to a powder-pattern-like sampling of orientations in the smaller amplitude high-field Pake doublet feature. Magnetic Field Dependence of the Experimental ESEEM. Figure 7 shows two-pulse ESEEM collected at the four magnetic field values of 306.5, 310.5, 316.5, and 322.0 mT. Each waveform is the quotient of the waveform from a 2H-labeled sample (dividend) and a nondeuterated (natural isotope abundance) sample (divisor). The quotient ESEEM only contains contributions from 2H nuclei exchanged onto C5′, and the 1H nuclei that were replaced.55,60 For the Bo range from 306.5 to 322.0 mT, the free 1H resonant frequency, νH, varies from 13.05 to 13.71 MHz and the free 2H resonant frequency, νD, varies from 2.00 to 2.10 MHz. The oscillations of shortest period near 72 and 36 ns are from the 1H matrix and sum-combination features, respectively, while the oscillations of longer period near 500 ns arise from 2H coupling. Prior to envelope division, the amplitude of each waveform decayed quasi-exponentially versus τ with a characteristic time given by Tm = 1.5 µs, the phase memory time. Therefore, the signal amplitude and signalto-noise is lowest for large τ, and this large relative noise at large τ is enhanced by the envelope division. Figure 7 also shows that the slightly more rapid phase memory decay in the protonated sample leads to a baseline drift of negative slope. This drift is accounted for by the C term in eq 12. The significant differences among the ESEEM collected at different magnetic fields shows that orientation selection is present. Figure 8 shows Fourier transforms of the ESEEM shown in Figure 7. The spectra are dominated by features from 2H hyperfine coupling. Significant intensity from 1H coupling

Canfield and Warncke

Figure 8. Fourier transforms of the two-pulse 2H/1H quotient ESEEM collected from the (S)-2-aminopropanol radical in ethanolamine deaminase at four different magnetic field values (solid lines), and corresponding overlaid simulations (dotted). Features arising from the strongly coupled 2H (2Hs) and the two weakly coupled 2H (2Hw) are noted. Experimental conditions: Same as those described in legend of Figure 7.

appears at νH (13-14 MHz) and 2νH (26-28 MHz), and is therefore not shown in the spectral range of Figure 8. Although the ESEEM spectra vary significantly with magnetic field, each line shape displays features at comparable frequency positions. As previously assigned,23 the single narrow feature at νD arises from two weakly coupled 2H nuclei. The pair of features that are split symmetrically about νD by approximately 1 MHz arise from the single strongly coupled 2H nucleus. The broad feature that stretches from below 3 MHz to 6 MHz arises from the weak ∆mI ) (2 frequencies, which occur at twice the corresponding fundamental frequencies. Very weak modulation at combinations of the fundamental frequencies61 is also present in this region. The negative phase sum-combination feature of the strongly coupled 2H nucleus is observed at 4.2-4.4 MHz. The shift of this feature from 2νD reflects the relatively large dipolar contribution to the hyperfine coupling.62 In our previous study of the substrate radical-C5′ methyl 2H hyperfine coupling, performed assuming a powder pattern distribution at 310.5 mT, we found that shifts of (0.1 Å from an r1 value of 2.2 Å led to dramatically poorer matches of the sum-combination feature position, showing the sensitivity of this feature to distance.62 These shifts in r1 also gave distortions in the fundamental region of the spectrum.23 In line with this, and to reduce the number of variable parameters, we have fixed the value of r1 to 2.2 Å in the present simulations. Inspection of the magnetic field dependence of the peak positions and line widths of the 2H features in the ESEEM spectra in Figure 8 provides qualitative insight into the orientation of the z-axes of the 2H hyperfine tensors (corresponding to the C1-2H axes) relative to B Bo. For the strongly coupled 2H, the maxima of the low and high-frequency features change from 1.54 and 2.54 MHz at 306.5 mT to 1.46 and 2.67 MHz at 316.5 mT. The splitting therefore increases from 1.0 to 1.2 MHz upon moving from the low field edge of the radical EPR line shape to the center of the line shape. This small but significant shift can be clearly observed in Figure 8 by reference to the dashed lines offset from νD by (0.5 MHz. Since the isotropic component of the hyperfine coupling of the strongly coupled

Reactant Center Geometry in Ethanolamine Deaminase TABLE 2. Best-fit ESEEM Simulation Parameters and Ranges Obtained from Global Fitting of the ESEEM Collected at Different Magnetic Field Valuesa variable 98 |C1-C5′|/Å 98 |CoII-C5′|/Åb C5′-H1-C1/deg Aiso,1/MHz r1/Å rb/Å rc/Å β1/deg βb/deg βc/deg

best-fit value

best-fit range

3.28

3.12-3.30

7.84 14.37 165 -0.29 2.20 3.85 3.87 1.77 12.5 14.9

7.81-8.25 14.07-14.39 139-175 -0.34 to -0.25 fixed 3.50-3.86 3.81-3.99 1.43-15.3 3.62-32.4 0.39-27.7

fit number

best-fit value

best-fit range

∑(1 - r )

0.349

0.349-0.357

2

a

The best-fit range column lists ranges from nearly 4000 separate fitting attempts (simulation runs). From the best-fit global fit number corresponding to the results in Figure 7, a best-fit range (listed at the bottom of the range column) was selected. Only fitting attempts with global fit numbers within this range were used to determine the ranges 98 for each parameter. Thus, if | C1-C5′| is in the range listed above, it is possible to obtain a global fit with a fit number within the best-fit range by using a combination of all the other fitting parameters from within their respective ranges. The βb, βc, rb, and rc parameters listed here are related to the β2, β3, r2, and r3 from Figure 3 but sorted so that rb e rc always holds. The complete fit number range and ranges for all parameters are shown in Figure S1. b Inversion symmetry of the model leads to two possible values for the distance.

is small relative to the dipolar component (|Aiso,1/Adip,1| ) 0.25) and negative, as described below, the powder pattern hyperfine line shape of this nucleus is characterized by features around νD ( A| /2, corresponding to the “A| region” of the hyperfine line shape (A| ) Aiso,1 + 2Adip,1), at the low- and highfrequency extrema and by features around νD ( A⊥/2, corresponding to the “A⊥ region” (A⊥ ) Aiso,1 - Adip,1), closer to νD. Therefore, the C1-2H vector is oriented approximately perpendicular to B Bo at 306.5 mT, and approximately along B Bo at 316.5 mT. These orientations of the electron-nuclear vector relative to B Bo are similar to the orientations of the electron-electron (CoII-C1) vector relative to the Bo vector at 306.5 and 316.5 mT. The same trends are observed for the feature at νD corresponding to the weakly coupled 2H. As shown in Figure 8, the line width of this feature is relatively narrow at 306.5 mT, but is broadened substantially at 316.5 mT. The approximate collinearity of the CoII-C1 and C1-2H vectors is borne-out by the P(θ) sin(θ)-weighted ESEEM simulations. Simulation of the ESEEM. Figure 7 shows that the quotient 2-pulse ESEEM simulations provide a very good match to the corresponding experimental waveforms. The parameter set used for the calculated ESEEM was obtained from global, or simultaneous, simulation of the ESEEM collected at the four magnetic field values. For these global fits, only five input pa98 rameters (|C1-C5′|, θ2, φ1, φ2, and Aiso,1) were varied. Best-fit 98 values and ranges for |C1-C5′| and Aiso,1, and derived atomatom distance and angular parameters, are presented in Table 2. The fitting parameters are presented in the legend to Figure 7. The origins of the parameter ranges are described in further detail and illustrated in the Supporting Information, Figures S1 and S2. The values of Aiso,1 ) - 0.29 MHz and r2, r3 values of 3.85, 3.87 Å are the same within error as the values of -0.35 MHz and 3.8 Å obtained in the previous 2H ESEEM study at a 2H

J. Phys. Chem. B, Vol. 106, No. 34, 2002 8839 single magnetic field value.23 The inversion symmetry of the β1 parameter in the coordinate system presented in Figure 3 leads to two possible values for the CoII-C5′ distance: 7.8 Å (corresponding to β1 ) 1.77 °) and 14.4 Å (corresponding to β1 ) 180-1.77 ) 178.2 °). These two distances are distinguished by additional considerations, described below. Finite and negative values of C reflect the slightly faster phase memory decay in the protonated sample. The A values of less than unity and positive B values for the fits shown in Figure 7 indicate that the extent of 2H coupling is less than expected on the basis of the assumed stoichiometry of two 2H nuclei per C5′ methyl group. We attribute the lower 2H incorporation to “washout” of the 2H label during substrate radical formation and cryotrapping, owing to limited turnover of the enzyme on the natural isotopic abundance 2-aminopropanol. The detailed analysis of this effect will be included in a subsequent report (J. Canfield and K. Warncke, manuscript in preparation). Figure 8 shows simulated Fourier transforms overlaid with the corresponding experimental Fourier transforms. Note that the experimental and simulated waveforms were processed in the same way to achieve the Fourier transforms, and that the ESEEM fitting procedure was performed on the waveforms, not the Fourier transforms. Although the match to the amplitudes is not in all cases exact, the simulations reproduce the broadening of the weakly coupled 2H and the increased splitting of the strongly coupled 2H features that are associated with the increase of the magnetic field from 306.5 to 316.5 mT. The relative amplitudes of the weakly coupled and the strongly coupled 2H features are also reproduced well, with the exception of the spectrum for 316.5 mT. The origin of the distortion of the FT, other than from waveform truncation effects, is not known. The muted difference between the maxima of the ESEEM line shapes corresponding to 306.5 mT and 316.5 mT is caused primarily by the modest angle selection afforded by the electron-electron dipolar interaction, and by two additional factors. First, the small Aiso,1 value of -0.29 MHz acts to move the A| and A⊥ turning points on each side of νD closer together. Second, the B Bo versus 98 C1-Hn orientation dependence of the ESEEM leads to zero amplitude at the canonical νD ( A| /2 and νD ( A⊥ /2 positions in the hyperfine line shape,53 unlike for the true electron-nuclear double resonance (ENDOR) line shape. Assessment of the Method. The method of simulating orientation selection in the radical component of the line shape of biradical systems in the regime of intermediate coupling strength that we present here bears strong similarities to the methods developed by Hoffman and co-workers for simulating the orientation-selected (polycrystalline) ENDOR in transition metal systems, where the line shape anisotropy is created primarily by anisotropy in the g-tensor.26-29 The ENDOR treatment elegantly and compactly references the crystallite populations to the g-factor [g(θ,φ), as portrayed on a unit sphere],26 whereas we describe the subpopulations directly in terms of θ,φ. The ENDOR method treats the finite line widths of both the EPR and ENDOR transitions by using isotropic Gaussian line shape functions.27 In our treatment of the biradical EPR spectrum, we also use isotropic Gaussian line shape functions [the line width parameter corresponds to the full width at 61% of the maximum] for the EPR lines. However, the width of the lines in the ESEEM spectrum is determined by the decay of the envelope modulation in the time domain. In the ENDOR simulations, a value of 0.2 MHz for the line shape function has been used,27 compared to the effective line width parameter in the ESEEM studies of 0.4 MHz, which is determined by the 2.4 µs length of the two-pulse waveform. Signal-to-noise

8840 J. Phys. Chem. B, Vol. 106, No. 34, 2002 considerations in two-pulse ESEEM, owing to the relatively short phase memory times, restrict the length of the time domain, and hence the effective fwhm of the intrinsic line widths in the FT. We have made approximations to simplify our treatment, which might affect the application of the method to systems of arbitrary g1, g2 values and electron-electron coupling strength. Hyperfine anisotropy for the organic radical is not explicitly incorporated in the EPR simulations, because the absence of resolved hyperfine coupling in the line shape does not allow us to accurately determine the hyperfine coupling tensors. For systems in which the anisotropic hyperfine coupling approaches the value of the electron-electron dipolar coupling, D, the line shape anisotropy will be determined by both interactions. Based on the reported structure,11 the substrate radical 2 includes a strong R-1H interaction. To assess the influence of strong, anisotropic hyperfine coupling, simulations were performed with coupling and line width parameters varied as before, but with an explicitly incorporated R-1H hyperfine interaction.50 Cases of the principal hyperfine axis oriented parallel and perpendicular to the CoII-C1 axis were considered. The resulting population [P(θ) or P(θ) sin (θ)] versus θ plots were found to be essentially the same as those in Figure 6 (Supporting Information, Figure S3). The line width parameter decreased, as expected from the additional line-broadening contribution of the hyperfine interaction, from 4.9 to 4.0-4.1 mT. These results support the use of the Gaussian EPR line shape function. The bandwith of the microwave pulse has also not been explicitly taken into account. Square π/2 pulse widths of 20 ns were used in this study, because of the requirement to excite a sufficient number of transitions to maintain adequate signalto-noise. The bandwidth (fwhm) of the two-pulse ESEEM experiment performed with 20 ns π/2 pulses is equivalent to 1.4 mT.63 Since 1.4 mT is approximately three-fold less than the fwhm of the line shape function, it appears that the neglect of the pulse bandwidth is reasonable. Finally, although the application of an S ) 1/2 Hamiltonian to describe the ESEEM from the substrate radical component of the relatively weakly coupled CoII-radical pair appears to be a good first-order approximation,9,49,64 subtle effects on the ESEEM intensities cannot be ruled out. We are currently developing a full-matrix approach for simulating ESEEM from biradical systems. Model for Geometry of Reactant Centers in the Active Site. Figure 9 shows the geometry of C1, CoII, C5′, and the three C5′ methyl hydrogen nuclei that is obtained from the best global fit parameters. The CoII-C1 distance is 11.1 Å, as obtained from the EPR simulations alone. For the model in Figure 9, we have chosen β1 ) 1.77 °, which places C5′ between CoII and C1. This choice is supported by X-ray crystallographic structures of the coenzyme B12-dependent enzymes diol dehydrase, glutamate mutase, and methylmalonyl-CoA mutase, which indicate that the region above the β-face of the cobalamin, between cobalt and the substrate reactant center, is relatively free, whereas the substrate is bound to the wall of a β8R8- (TIM-) barrel structure, and space on the side of the substrate opposite the CoII is filled by protein.65 Mechanistic proposals that are based on the X-ray structures show C5′ shuttling directly between cobalt and the substrate.65b,c,f,g Thus, in our model, C5′ is positioned at distances of 3.3 Å from C1 and 7.8 Å from CoII. This positioning places C5′ essentially along the CoII-C1 axis. The C1-H-C5′ angle for the strongly coupled hydrogen is 165°. The deviation of this angle from linearity is not significant to within the ranges of fitting parameters. As inferred from the inspection of the dependence of the hyperfine features

Canfield and Warncke

Figure 9. Model for the structure of the reactant centers in the active site of the CoII-substrate radical pair state in ethanolamine deaminase. The positions of the CoII, C1, C5′, and C5′ methyl hydrogen atoms are marked by small spheres. The positions and orientations of the atom centers are to scale, relative to the plane of the CoII-C1 axis, which lies in the plane of the page. The distance scale is in the plane of the CoII-C1 axis. (A) View along line perpendicular to CoII-C1 axis with C5′-C1 axis eclipsed. (B) View after 90° rotation about the CoII-C1 axis, relative to (A).

on magnetic field, the CoII-C1 and C1-2H axes in this model are approximately collinear (βn e 14.9° for all Hn). Implications for the Mechanism of Catalysis. The orientation-selection-ESEEM results confirm our previous conclusion that the C5′ center in the 5′-deoxyadenosyl radical directly abstracts the hydrogen atom from the substrate, and extends the earlier conclusions by allowing the position of C5′ to be specified relative to C1 and CoII. The orientation-selection results establish that C5′ undergoes a migration of 6 ( 1 Å from the bonded position = 2.0 Å from the cobalt atom in adenosylcobalamin,8 to the observed position in the substrate radical state. The displacement of 6 ( 1 Å is significantly greater than the difference of 1.3-1.4 Å between the C5′ positions in intact adenosylcobalamin and in 5′-deoxyadenosine bound in the active site of glutamate mutase,65g a Class I adenosylcobalamindependent enzyme. The radical migration mechanism is therefore different in ethanolamine deaminase and glutamate mutase. Along with the EPR-determined radical pair distances66 and the X-ray crystallographic structures, these results suggest that the distance that the radical migrates is a general feature that distinguishes the Class I and II enzymes. The reactant center positioning in Figure 9 suggests that the coordinate for C5′-mediated radical migration lies very close to the CoII-C1 axis. The model also suggests that the coordinate for hydrogen atom transfer lies close to the CoII-C1 axis. Following hydrogen atom transfer, which probably occurs at a C1-C5′ separation of < 3.0 Å, the C5′ group retracts from C1 to its observed position, 3.3 Å distant. This retraction may be a mechanistic feature that deters radical pair recombination. The near collinearity of the cobalt-carbon bond axis in the intact coenzyme, the radical migration coordinate, and the hydrogen atom transfer coordinate suggests an economy in the molecular motion within the active site that would minimize rate-slowing molecular reorganization associated with the long-range radical pair separation, and also allow less opportunity for interaction of the unstable C5′ radical with the surrounding protein. Acknowledgment. Supported by grant R01 DK54514 from the National Institutes of Health. We thank Ms. Lori Anderson for technical assistance and Professor Dale E. Edmondson (Emory University) for use of fermentation facilities.

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