High-Frequency Electron Paramagnetic Resonance Spectroscopy of

Yuk-Ki Lee , Mei M. Whittaker and James W. Whittaker .... Alexandre M Boulet , Eric D Walter , Dwight A Schwartz , Gary J Gerfen , Patrik R Callis , D...
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J. Phys. Chem. 1996, 100, 16739-16748

16739

High-Frequency Electron Paramagnetic Resonance Spectroscopy of the Apogalactose Oxidase Radical Gary J. Gerfen, Brendan F. Bellew, and Robert G. Griffin* Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

David J. Singel Department of Chemistry and Biochemistry, Montana State UniVersity, Bozeman, Montana 59717

Christopher A. Ekberg and James W. Whittaker* Department of Chemistry, Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000 ReceiVed: March 7, 1996; In Final Form: July 17, 1996X

The activated form of galactose oxidase from the fungus Dactylium dendroides contains a single divalent copper ion which is antiferromagnetically coupled to a protein-based free radical. Chemical oxidation of the apoenzyme generates the free radical which is localized on a covalently cross-linked tyrosine-cysteine residue. This species, together with model radicals generated by UV irradiation of protonated and selectively deuterated o-(methylthio)cresol (MTC), has been studied by high-frequency EPR spectroscopy (139.5 GHz/5 T) in conjunction with molecular orbital calculations employing self-consistent local density functional (LDF) methods. The Zeeman interactions (g values) determined from the high-frequency spectra of the apogalactose oxidase and the MTC model radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbital calculations accurately reflect the experimental data, including an increase in the axial symmetry of the Zeeman interaction for the MTC radical compared with the unsubstituted tyrosyl radical species. An explanation of this effect based on an analysis of individual atomic contributions to the molecular g values is presented. High-frequency echo-detected EPR spectroscopy of the apogalactose oxidase radical resolves hyperfine splittings. Based on the molecular orbital calculations and the EPR spectroscopic results presented here, the hyperfine splittings are assigned to two methylene protonssone derived from tyrosine and one from cysteine. These findings are consistent with the radical spin density being localized on the tyrosine-cysteine moiety, rather than delocalized throughout an extended π-network involving a nearby tryptophan as had been previously suggested as a possible explanation of the stability of the radical species.

Introduction Galactose oxidase from the fungal species Dactylium dendroides is a monomeric 68 kDa metalloenzyme catalyzing the two-electron oxidation of a broad range of primary alcohols to the corresponding aldehydes, with the concurrent reduction of O2 to H2O2.1-3 A single Cu ion is bound to the protein as the sole metal cofactor. The enzyme is activated by one-electron oxidation, leading to the disappearance of the cupric EPR (electron paramagnetic resonance) spectrum characteristic of the native enzyme and to the formation of an oxidized copper complex with no observable (X-band) EPR spectrum.4,5 Optical and X-ray absorption measurements have demonstrated that the metal remains divalent upon oxidation of the complex,5-7 implying the generation of a free radical at some protein redox site. Magnetic susceptibility experiments on the activated enzyme reveal a strong antiferromagnetic exchange coupling |J| > 200 cm-1 for the radical-copper complex.3,8 This feature defines a new class of radical-copper oxidases containing an antiferromagnetically coupled free radical-copper complex that serves as a two-electron redox unit in the active enzyme. The protein free radical in galactose oxidase can be prepared in up to 40% yield by mild oxidation of the apoprotein formed by removal of copper.8 The distinctive EPR spectrum of this * Authors to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, September 15, 1996.

S0022-3654(96)00709-5 CCC: $12.00

stable radical species (half-life at 273 K on the order of weeks) is significantly perturbed by the incorporation of methylene[β,β-2H]-deuterated tyrosine into the enzyme, indicating that the radical involves a tyrosyl residue.8 An X-ray crystal structure of galactose oxidase has recently revealed two tyrosines coordinated to the active site copper ion.9 One of these tyrosines (Y272) is covalently cross-linked to a cysteinyl residue (C228), with the sulfur of the cysteinyl side chain replacing hydrogen on one of the ortho ring carbons. EPR and ENDOR (electron nuclear double resonance) experiments have identified this novel modified tyrosine as the protein free radical site in apogalactose oxidase.10 Tyrosyl radicals have been identified and studied in the structures of an array of enzymes,11 including ribonucleotide reductases,12-14 prostaglandin synthase,15,16 and photosystem II.17-21 (Under certain conditions, cytochrome c peroxidase appears to form a tyrosyl radical, but its exact location and function remain unknown.22) The widespread occurrence of tyrosyl radicals in biology is likely a consequence of the relatively low redox potential of the phenolic side chain. However, radicals have recently been identified or implicated at other sites in proteins, including glycine,23 tryptophan,24 and cysteine25,26 residues. There is a widening recognition of the importance of radicals in biochemistry leading to the emergence of a new field of free radical enzymology.27,28 The free radical in galactose oxidase is of special interest in this growing list of © 1996 American Chemical Society

16740 J. Phys. Chem., Vol. 100, No. 41, 1996 biological radicals in that it directly participates in active site chemistry as a functional redox site,3,5,29,30 in contrast to many other protein radicals that appear to store redox equivalents and are only indirectly involved in catalytic function. The importance of free radicals in enzyme catalysis has led to the development of effective spectroscopic probes to study protein-based radicals. Optical absorption and resonance Raman spectroscopies have proven useful in identifying radicals based on characteristic spectral features but give only limited information on the details of electronic and molecular structure. EPR, ENDOR, and ESEEM (electron spin echo envelope modulation) spectroscopies31,32 have proven especially valuable in studies of radicals as a result of both their selectivity for the paramagnetic species and their sensitivity to details of the ground-state electron spin distribution. This spin distribution, which can be characterized through the Zeeman and electron-nuclear hyperfine interactions,33 directly reveals the nature of the electronic wave function involved in redox chemistry. These advantages of EPR methodologies have been extensively developed in numerous low-frequency investigations and have been valuable in identifying and characterizing protein radicals.14,21,24,34,35,36 High-frequency EPR spectroscopy is a technique that can extend the information available from conventional lowfrequency methods in studies of biological free radicals.37-44 These radicals are typically studied in frozen solutions, and as such their EPR spectra consist of overlapping powder patterns arising from anisotropy in both the electron Zeeman interaction (g values) and electron-nuclear hyperfine interactions. At conventional field strengths (∼0.3 T), interpretation of these spectra can be complicated since the spectral dispersion arising from the Zeeman anisotropy is similar to that spanned by hyperfine interactions. At high fields (∼5 T), however, the Zeeman anisotropy tends to dominate the spectrum. Consequently, accurate g values are readily obtained, and any hyperfine splittings observed can be related to the Zeeman axis frame. These experimentally determined parameters, in conjunction with theoretical analyses provided by molecular orbital calculations, can furnish detailed information concerning electronic wave functions. In particular, it is possible to characterize electron spin density at atomic centers such as oxygen and sulfur in natural abundance. Because the predominant isotopes of these nuclei lack magnetic moments, this information is difficult to obtain from hyperfine structure without the substitution of magnetically active isotopes. This study uses a combination of high-frequency EPR spectroscopy and molecular orbital calculations to investigate and compare the structures (Figure 1) of the novel tyrosinecysteine radical in apogalactose oxidase both with a model radical generated by UV photolysis of o-(methylthio)cresol (MTC) and with the tyrosyl radical found in the R2 subunit of E. coli ribonucleoside diphosphate reductase (RDPR). The 139.5 GHz pulsed (echo-detected) EPR spectroscopy of the apogalactose oxidase radical resolves proton hyperfine couplings and relates them to the Zeeman principal axis system. EPR spectroscopy of both the apogalactose oxidase and the model compound (methylthio)cresyl radical species reveals a significant perturbation of the ground-state electronic structure induced by thioether substitution. Molecular orbital calculations trace this perturbation to a heavy atom effect arising from the spin-orbit coupling associated with the sulfur-containing side chain. EPR spectra of the galactose oxidase and MTC radicals both exhibit nearly axial symmetry for the Zeeman interactions, in contrast to the distinctly rhombic Zeeman interactions typical of tyrosine phenoxyl radicals in RDPR39 and other proteins,45 as well as in irradiated single crystals of L-tyrosine.46 Electronic structure calculations of the spin-unrestricted ground state of the MTC

Gerfen et al. phenoxyl radical using local density functional (LDF) methods have allowed a calculation of the molecular g matrices for comparison with experiment. The axial g matrices observed for the sulfur-substituted radicals result from the transfer of substantial unpaired spin density onto the exocyclic sulfur through π covalency in the highest occupied MO. This delocalization of the redox orbital onto sulfur may also contribute to the lowering of the redox potential for thioethersubstituted phenoxyl radicals, relative to their unsubstituted counterparts, by as much as 0.5 V.47,48 Spin density distributions determined from these LDF-MO calculations lead us to assign the hyperfine splittings observed in the apogalactose oxidase radical spectra to two methylene protonssone derived from tyrosine and one from cysteine. Spectra and simulations of o-(methylthio)cresyl radical deuterated at the thiomethyl position provide compelling support for this assignment, which is distinct from that made in the lowfrequency ENDOR study of this radical species.10 This previous study attributed both hyperfine interactions to protons on tyrosinesone to a β methylene and one to a ring proton ortho to the oxygen. Materials and Methods Samples. Apogalactose oxidase was prepared and the radical species generated by ferricyanide treatment as previously described.8 The concentrations of protein and radical in the samples were 1.3 and 0.16 mM, respectively. o-(Methylthio)cresol (MTC) was prepared as previously described49 and fractionally distilled under vacuum before use. The o-(methylthio)cresol samples were prepared as 2 mM solutions in propionitrile:butyronitrile glassing solvent (1:1 molar ratio, from freshly distilled components)50 and deprotonated with a stoichiometric equivalent of tetrabutylammonium hydroxide. The thiomethyl-deuterated compound was synthesized by a modification of the published procedure49,51 using (methyl sulfide)d6. The 1H-NMR spectrum of the product was identical to that of protonated MTC, except for the absence of the δ ) 2.34 ppm (-S-CH3) resonance. Samples were sealed in glass ampules under argon, shipped on dry ice, and stored in liquid nitrogen. Immediately prior to each EPR experiment, the ampules were thawed and broken, and the liquid was taken up into quartz capillary tubes (0.4 mm i.d., 0.55 mm o.d.). Radicals of the MTC model compound were generated by UV irradiation (lamp, UVP Model B 100 AP; bulb, Sylvania Model H44GS-100, 100 W) of the loaded sample tubes under liquid nitrogen. Irradiation time of displayed spectra of the o-(methylthio)cresol was approximately 1 min. At longer irradiation times, the EPR spectra of the thioether derivative were found to be highly variable, appearing to reflect photochemical decomposition of the sample. The estimated concentration of the UV-generated radical species is 10-50 µΜ. The samples of RDPR (200 µΜ in tyrosyl radical) were prepared as previously described.52 139.5 GHz EPR Spectroscopy. EPR spectra were acquired in continuous wave (CW) unsaturated absorption, CW saturated dispersion, and electron spin-echo modes. High-frequency spectra were obtained using a 139.5 GHz heterodyne spectrometer with phase-sensitive detection designed and fabricated in this laboratory.53,54 Samples contained in quartz capillary tubes were inserted into a cylindrical TE011 resonator under liquid nitrogen, and the EPR probe was loaded into a precooled cryostat as previously described.55 A steady flow of cold helium gas over the cavity provided sample cooling during the experiments. In order to increase signal-to-noise ratios, a set of spectra were obtained under conditions which maximized the saturated

EPR of Apogalactose Oxidase Radical

J. Phys. Chem., Vol. 100, No. 41, 1996 16741

first harmonic (modulation-detected) dispersion signal. Typical saturating conditions were as follows: microwave power at the sample ∼20 µW corresponding to a B1 field of ∼0.01 mT; temperature 10 K, modulation amplitude and frequency equal to 0.24 mT and 400 Hz, respectively. Saturation and adiabatic passage effects in high-frequency EPR spectroscopy have been discussed previously.42,45,55 The modulation-detected dispersion signals have an unconventional appearance and exhibit increased line broadening (and consequently decreased spectral resolution) compared to conventional, unsaturated absorption EPR signals. In order to achieve a more conventional presentation, saturated dispersion spectra were subjected to pseudomodulation methods developed by Hyde et al.;56 the algorithm also has the effect of acting as a digital noise filter.56 When signal-to-noise ratios allowed, the unsaturated absorption mode and echo-detected EPR (EDEPR) techniques were employed in an effort to detect proton hyperfine structure not resolved in the saturated dispersion spectra. For the highfrequency echo-detected EPR spectroscopy,53 the microwave power was approximately 800 µW, corresponding to a B1 field of ∼0.06 mT. A three-pulse echo sequence (p1-τ-p2-T-p3) was employed, with pulse widths (p) of 0.35 to 1.5 µs, interpulse delays (τ, T) of 0.3-2 µs, and repetition rates of 100 Hz. The echo was acquired at a time τ after the third pulse by a boxcar integrator (E.G.&G. Princeton Applied Research). To facilitate comparison with CW spectra, the echo-detected EPR spectra were also subjected to psuedomodulation. The external magnetic field was typically swept over a 25 mT range at 0.01 mT/s. The field was calibrated by recording the EPR spectrum of Mn0.0002Mg0.9998O (ge ) 2.001 01 and AMn ) 8.71 mT) under conditions identical to those used for obtaining the EPR spectrum of the sample. Resonant field values of the MnO standard were then compared to calculated values following the procedure of Burghaus et al.57 to determine the magnitude of the field corrections. Spectral Simulations. All EPR spectra were simulated, as previously described,55 with the following adjustable parameters: the three principal values of the g matrix, the three principal values of each hyperfine interaction matrix; and the orientation of the principal axes of each hyperfine matrix within the Zeeman interaction principal axis system. The resonant fields (Bres) were determined using the expression

Bres(θ,φ,m I1,...,m In) )

pωe g(θ,φ)β

n

- ∑m Ii Ai(θ,φ)

(1)

i)1

in which θ is the polar angle and φ is the azimuthal angle that together specify the orientation of the external magnetic field B0 in the Zeeman principal axis system, p is Planck’s constant, ωe is the microwave angular frequency, g(θ,φ) is the orientationally dependent g value, β is the Bohr magneton, and Ai(θ,φ) (in millitesla) is the orientationally dependent hyperfine coupling to the ith nucleus. In the simulation of orientationally disordered samples 180 polar angles were sampled at equal intervals over a range of 0 to π. At each polar angle, between 1 and 360 azimuthal angles (the number being nearly equal to 360 times the sine of the polar angle) were sampled at equal intervals over a range 0 to 2π. At each orientation the resonant fields were calculated, and the resulting distribution of resonant fields was convolved with the appropriate line shape. This line shape function was a Gaussian curve for echo-detected spectra and the derivative of a Gaussion for CW unsaturated first harmonic absorption spectra. For CW saturated disperion spectra (in phase with the modulation), the calculated spectra were convolved with a function described by Ammerlann et al.,58 which we have found accurately reproduces our experi-

mentally observed line shapes. The simulations were then pseudomodulated56 in a manner identical to the experimental data in order to display the spectra in a more conventional “derivative” mode. In order to assess the validity of the first-order treatment, we carried out additional simulations employing the exact treatment of the proton spin Hamiltonian with the largest degree of anisotropy in its hyperfine interaction. Simulations based on this more exact treatment, which calculates the resonant field and transition probability of each “allowed” and “forbidden” EPR transition, were virtually indistinguishable from spectra calculated with eq 1. Molecular Orbital Calculations. Calculations were performed using self-consistent local density functional (LDF) methods59 implemented in the program DMol (Biosym Technologies, San Diego). DMol uses the Vosco-Wilk-Nusair exchange-correlation potential60 with Becke 88 exchange61 and Lee-Yang-Parr (LYP) correlation nonlocal corrections,62 providing a first principles calculation of electronic structures of molecular systems. For large molecular systems LDF methods offer significant advantages in computational efficiency and are also less susceptible to spin contamination problems than Hartree-Fock calculations.63-65 The calculations were performed using a double numeric basis extending to the third harmonics for all non-hydrogen atoms which allows for adequate treatment of polarization effects. Molecular geometries were optimized by relaxing the structures in spin-unrestricted, correlated ab initio potentials. During geometry optimization, significant eigenvector mixing (0.3) was allowed while stricter convergence criteria (0.005 mixing coefficients for electron density and spin) were required to obtain high-quality groundstate wave functions. This was particularly important for calculation of electronic structure of the MTC radical, for which low-energy electronic excited states are predicted. The spinunrestricted ground-state electronic structure was solved in the optimized geometry. The resulting molecular orbital wave functions were expanded in atom-centered numerical basis sets to obtain the coefficients used in computing g values. Calculations were performed on a Silicon Graphics Power Indigo2 XZ workstation with a R8000 processor running under a 64-bit Irix 6.0 operating system. Graphical displays were printed from the Insight ΙΙ molecular modeling interface (Biosym Technologies, San Diego). The unpaired spin distribution over the entire molecular structure determines the ground-state magnetic properties of the radical species. For the compounds of interests, which exhibit Cs point symmetry, all electronic wave functions are nondegenerate and in the absence of spin-orbit coupling: the orbital momentum is quenched.66 In this low-symmetry limit, spinonly paramagnetism is expected, resulting in an isotropic Zeeman interaction governed by the free electron g value (ge). The effect of spin-orbit coupling is to mix orbitals in the a′ irreducible representation of Cs with a′′ orbitals Via transverse components of orbital angular momentum (LX, LY). The consequent “unquenching” of orbital angular momentum is reflected by EPR g shifts defined as ∆g ) g - ge1, in which g is the g matrix, 1 is the unit matrix, and ge ) 2.002 32 is the free electron g value. g values can be calculated from the molecular orbital description of a radical by a sum-over-states approach in second-order perturbation theory for the combined Zeeman and spin-orbit perturbations.67,68 The spin-orbit operator is taken as a single-center operator, as is typically done in calculations of π systems containing oxygen or sulfur.69-71 The results of this study (Vide infra) confirm that single center terms from oxygen and sulfur dominate the g shifts and justify the neglect of multicenter terms. The g shifts can thus be written

16742 J. Phys. Chem., Vol. 100, No. 41, 1996

Gerfen et al. (j,k′) (i,k′) (i,k) (j,k) ∆gXX ) 2∑∑R* RY ∑ζ(k)R* RZ /(i - j) (3a) Z Y i*j k′

k

(j,k′) (i,k′) (i,k) (j,k) RY ∑ζ(k)R* RZ /(i - j) (3b) ∆gXY ) -2∑∑R* Z X i*j k′

k

(j,k′) (i,k) (j,k) RX(i,k′)∑ζ(k)R* RZ /(i - j) (3c) ∆gYY ) 2∑∑R* Z X i*j k′

Figure 1. Schematic representation of the molecules under study. The molecule-based axis system used in the molecular orbital calculations is denoted in upper case letters, while the principal axes for the proton hyperfine interactions are given in lower case letters.

as a sum of contributions from the individual atoms in the molecule, with each contribution involving matrix elements of orbital angular momentum operators between the unperturbed molecular orbitals:67,68

∆gXX ) 2∑∑〈ψ(j)|LX(k′)|ψ(i)〉∑ζ(k)〈ψ(i)|LX(k)|ψ(j)〉/(i - j) i*j k′

k

(2a)

These equations describe the total molecular g shift for the radical. It is also instructive to use eqs 3 in the determination of the individual contribution made by each atom to the molecular g matrix. The contributions to the g shifts along the X and Y axes for the kth atom are given by k (j,k′) (i,k′) (k) (i,k) (j,k) ) 2∑∑R* RY (ζ R* RZ )/(i - j) (4a) ∆gXX Z Y i*j k′

k (j,k′) (i,k) (j,k) ) 2∑∑R* RX(i,k′)(ζ(k)R* RZ )/(i - j) (4b) ∆gYY Z X i*j k′

These elementary g shifts are defined in a uniform coordinate system (X,Y,Z as illustrated in Figure 1), and each component of the atomic contributions combine to form the total molecular g shift: k ∆gQQ′ ) ∑∆gQQ′

∆gXY ) 2∑∑〈ψ |LX |ψ 〉∑ζ 〈ψ |LY |ψ 〉/(i - j) (j)

(k′)

(i)

i*j k′

(k)

(i)

(k)

(j)

k

(2b)

∆gYY ) 2∑∑〈ψ |LY |ψ 〉∑ζ 〈ψ |LY |ψ 〉/(i - j) (j)

(k′)

(i)

i*j k′

(k)

(i)

k

(k)

(j)

(2c)

in which i indexes MO’s and j indexes the singly occupied molecular orbital (SOMO), k and k′ index atoms, i is the eigenvalue of the ith MO, and ζ(k) is the one-electron atomic spin-orbit coupling constant for the kth atom (ζC ) 29.0 cm-1, ζO ) 147cm-1, ζS ) 374 cm-1).72 The coordinate system for the two radicals in Cs symmetry is defined by the normal to the aromatic ring (Z axis). The X and Y axes may be taken as any two directions mutually perpendicular to the ring normal forming a right-handed coordinate system. Results of calculations performed in a frame associated with an arbitrary choice of X and Y directions can be converted to a common representation placing the X axis along the phenoxyl C-O bond vector by a similarity transformation (Figure 1). Note that ∆gYX ) ∆g*XY and ∆gZZ ) 0 in Cs symmetry. The molecular orbitals can be expanded in terms of atomic valence orbitals,

|ψ(j)〉 ) ∑RZ(j,k)|2pZ(k)〉 k

|ψ(i)〉 ) ∑Rs(i,k)|2s(k)〉 + RX(i,k)|2pX(k)〉 + k

RY(i,k)|2pY(k)〉 + RZ(i,k)|2pZ(k)〉 In calculating the g shifts, the summations in eqs 2 are restricted to the MO’s within 0.5 hartree of the singly-occupied molecular orbital; outside of this set the contributions to the g shifts become insignificant because of the large energy difference denominators (Vide infra). This restriction, together with the approximation that only one-center contributions to the matrix elements of LX,Y need be considered, leads to the following convenient expressions for the g shifts in terms of the atomic orbital coefficients in the linear combination of atomic orbitals (LCAO) expansion of the MO’s:

k

(5)

k

Using this approach, the complete g matrix is calculated as g ) ge 1 + ∆g and is diagonalized to determine its principal values and the projection of the eigenvectors (principal axis orientations denoted by 1, 2, 3) in the molecule-based (X, Y, Z) axis system. In the cases considered here the g1 and g2 principal axes lie in the molecular plane and are uniquely defined by a single angle (Γ) with respect to the X axis. A copy of the source code for the program “g-val” used in computing the g shifts for molecular radicals may be obtained by submitting an e-mail request to [email protected]. Results EPR Spectroscopy. Figure 2 displays the low-frequency (9 GHz) EPR spectra of the apogalactose oxidase (A) and o-(methylthio)cresyl radicals (C), as well as the tyrosyl radical from E. coli ribonucleoside diphosphate reductase (E). The X-band EPR spectra of these species, which have been previously described,8,10,12,14 are reproduced here along with simulations (B, D, and F) for comparison with high-frequency spectra. These X-band simulations were computed with parameters listed in Table 1. Anisotropy in the g matrix (∼0.006) gives rise to spectral dispersions of ∼2.0 mT at fields corresponding to 9 GHz excitation (0.3 T). Since this dispersion is similar to that spanned by the proton hyperfine interaction, the determination of g values is complicated in these randomly oriented (frozen solution) samples at X-band. Figure 3 displays high-frequency (139.5 GHz) CW EPR spectra and simulations corresponding to those of Figure 2. In contrast to spectra obtained at 9 GHz, the high-frequency spectra are dominated by dispersion arising from g anisotropy (∼30 mT) at these higher field strengths (5 T); the precise determination of principal g values is thus facilitated (Table 1). The depicted spectra are either pseudomodulated saturated dispersion spectra (A) or absorption spectra (C and E). Simulations displayed in Figure 2 (B, D, and F) were computed using the parameters in Table 1. The hyperfine splittings clearly evident in the 9 GHz spectra of the apogalactose oxidase radical (Figure 2A) do not appear

EPR of Apogalactose Oxidase Radical

J. Phys. Chem., Vol. 100, No. 41, 1996 16743 TABLE 1: Principal Values Used in Simulationsa g1 g2 g3 A xβ7 A yβ7 A zβ7 A x′β8 A y′β8 A z′β8

apogalactose oxidase

MTC

2.00741b 2.00641b 2.00211b 1.55d 1.42d 1.42d 0.95e 0.80e 0.85e

2.00720b 2.00620b 2.00190b 0.92f 0.92f 0.92f 0.50f,g 0.50f,g 0.50f,g

RDPR g1 g2 g3 A xR3,5 A yR3,5 A zR3,5 A x′β7 A y′β7 A z′β7

2.00912c 2.00457c 2.00225c 1.05h 0.27h 0.79h 2.07d 1.98d 1.87d

a Absolute values of hyperfine principal values (in mT) are listed. See also refs 10, 14, and 39. Estimated uncertainties in g values are approximately 0.000 05. b Principal axis orientations shown in Figure 6F. c Principal axis orientations shown in Figure 6E. d Principal axis orientations collinear with those of the Zeeman interaction. e Principal axis corresponding to A z′β8 collinear with g3 and A x′β8 rotated from g1 by 20° (approximately along C-O bond). f Three equivalent protons with these couplings were used in the simulations. g These values were multiplied by 0.154 to simulate the substitution of protons (spin 1/2) with deuterons (spin 1) for simulation displayed in Figure 5. h Two protons with these couplings and with principal axis orientations shown in Figure 1 were used in the simulations.

Figure 2. X-band EPR spectra of oxidized apogalactose oxidase (A), UV-irradiated o-(methylthio)cresyl (MTC) (C), and the tyrosyl radical from the R2 subunit of E. coli ribonucleoside diphosphate reductase (RDPR) (E). Simulations (B, D, and F) are calculated with parameters given in Table 1. (A) EPR spectrum of apogalactose oxidase acquired with the following parameters: microwave frequency and power, 9.220 GHz and 2 µW, respectively; temperature, 8.2 K; time constant, 0.5 s; modulation amplitude, 0.02 mT. (B) Simulation of (A) using Gaussian line broadening of 0.57 mT. (C) EPR spectrum of MTC acquired with the following parameters: microwave frequency and power, 9.439 GHz and 2.5 µW, respectively; temperature, 10 K; time constant, 0.3 s; modulation amplitude, 0.1 mT. (D) Simulation of (C) using Gaussian line broadening of 0.49 mT. (E) EPR spectrum of RDPR acquired with the following parameters: microwave frequency and power, 9.057 GHz and 1 mW, respectively; temperature, 110 K; time constant, 32 ms; modulation amplitude, 0.2 mT. (F) Simulation of (E) using Gaussian line broadening of 0.80 mT.

in the pseudomodulated saturated dispersion spectra obtained at 139.5 GHz (Figure 3A). This raises the question of whether the lack of resolution results from line broadening introduced by saturation or from inherent spectral line widths (both homogeneous and inhomogeneous) at 5 T. To address these issues, we obtained echo-detected EPR spectra (Figure 4) using pulsed techniques previously described.53 The echo-detected EPR spectrum (Figure 4A), obtained with low-power excitation (B1 ∼ 0.06 mT), exhibits drastically reduced line broadening relative to the saturated dispersion spectrum (Figure 3A) and yields increased resolution of the proton hyperfine splittings, particularly at the high-field turning point. Echo-detected EPR spectroscopy offers a convenient means to resolve hyperfine splittings in situations where saturation-induced line broadening of CW spectra prevents their observation. Figure 5 presents X-band EPR spectra of UV-irradiated protonated MTC (A, B) and thiomethyl-deuterated (-S-CD3) MTC (C,D) acquired at 100 K. The simulation of the selectively deuterated species (D) was calculated using Hamiltonian parameters identical to those used for protonated MTC (B), except for the substitution of deuterons for protons at C8 (Table 1). Further details concerning the hyperfine principal values

Figure 3. Continuous wave 139.5 GHz EPR spectra of apogalactose oxidase (A), o-(methylthio)cresyl (MTC) (C), and the tyrosyl radical from the R2 subunit of E. coli ribonucleoside diphosphate reductase (RDPR) (E). Simulations (B, D, and F) were calculated using parameters listed in Table 1. (A) Saturated dispersion signal (subsequently subjected to pseudomodulation) of apogalactose oxidase acquired with the following parameters: microwave power, 20 µW; temperature, 10 K; time constant, 1 s; modulation amplitude, 0.24 mT. (B) Simulation of (A). The calculation simulates the saturated dispersion line shape of the acquired spectrum (with an underlying Gaussian line width of 1.0 mT) which was then subjected to the same pseudomodulation algorithm as was the experimental data. (C) Unsaturated modulation-detected absorption EPR spectrum of o-(methylthio)cresyl (MTC) radical acquired with the following parameters: microwave power, 10 µW; temperature, 12 K; time constant, 1 s; modulation amplitude, 0.24 mT. (D) Simulation of (C) using a Gaussian line broadening of 1.0 mT. (E) Unsaturated modulation-detected absorption EPR spectrum of RDPR acquired with the following parameters: microwave power, 15 µW; temperature, 90 K; time constant, 1 s; modulation amplitude, 0.24 mT. (F) Simulation of (E) using a Gaussian line broadening of 0.82 mT.

and assignments are given in the Discussion section. The g values used for each simulation displayed in Figures 2-5 were measured directly from the high-frequency spectra. The hyperfine interaction principal values listed in Table 1 were determined from 9 GHz EPR spectra (Figures 2 and 5), ENDOR

16744 J. Phys. Chem., Vol. 100, No. 41, 1996

Gerfen et al.

Figure 4. (A) Echo-detected 139.5 GHz EPR spectrum of apogalactose oxidase acquired at 10 K with 800 µW of microwave power incident on the cavity. Other experimental parameters as given in the text. The experimentally acquired EDEPR spectrum was subsequently pseudomodulated. (B) Simulation of (A) using an orientationally dependent Gaussian broadening function (with principal values of 0.90, 0.85, and 0.70 mT corresponding to principal axes of g1, g2, and g3, respectively). The simulated spectrum was subjected to the same pseudomodulation algorithm as was the experimental data.

Figure 6. (A) Half-occupied molecular orbital of the p-cresyl radical contoured to 0.55 e/Å3 using self-consistent local density functional methods. (B) Half-occupied molecular orbital of the o-(methylthio)cresyl radical contoured to 0.55 e/Å3. (C) Molecular structure of the cresyl radical as determined by optimizing the molecular geometry. Values for R1, R2, and R3 are 121.6°, 121.5°, and 115.0°, respectively. (D) Molecular structure of the (methylthio)cresyl radical. Values for R1, R2, and R3 are 121.6°, 122.2°, and 115.0°, respectively. (E) Calculated unpaired electron density at each atom and the orientation of the g principal axes for (E) the cresyl radical and (F) the o-(methylthio)cresyl (MTC) radical.

TABLE 2: Calculated Principal g Values and Principal Axis Orientations MTC radical cresyl radical Figure 5. X-band EPR spectra of UV-irradiated o-(methylthio)cresol (MTC) protonated (A, B) and deuterated at the C8 position (-S-CD3) (C,D). (A) Experimental spectrum of protonated MTC acquired with the following parameters: microwave frequency and power, 9.44 GHz and 1 mW, respectively; temperature, 101 K; time constant, 0.164 s; modulation amplitude, 0.03 mT. (B) Simulation using parameters listed in Table 1 (0.53 mT Gaussian line broadening). (C) Experimental spectrum of thiomethyl-deuterated MTC acquired with parameters identical to (A). (D) Simulation using parameters listed in Table 1 (0.70 mT Gaussian line broadening) with the substitution of three deuterons (0.077 mT isotropic hyperfine coupling) at the C8 position.

spectroscopic results,10,14 and the 139.5 GHz EPR spectra (Figures 3 and 4, ref 39). The E. coli RDPR tyrosyl radical, which has been studied previously using ENDOR14 and highfrequency EPR39 spectroscopies, is presented here for the purpose of comparison. Molecular Orbital and g Value Calculation. The model phenoxyl radicals used in the calculation to represent the apogalactose oxidase and RDPR radicals were taken as neutral species in Vacuo. The starting point for the geometry optimization was the Cs conformer having bond lengths and bond angles given by the default structural parameters of the Insight II modeling interface. For the model phenoxyl radicals used in the calculation to represent the apogalactose oxidase and RDPR radicals, the SOMO possesses a′′ symmetry and has the special significance of being the redox orbital of the radicals, defining their characteristic regiochemistry. For example, unpaired

g1

g2

g3

Γ, deg

2.006 05 2.017 04

2.004 36 2.003 35

2.002 32 2.002 32

23.58 0.02

electron density on the ortho and para ring carbons leads to characteristic carbon-carbon coupling reactions at these positions. In galactose oxidase, the π radical localized on the tyrosine-cysteine complex (Y272-C228) participates directly in the catalytic oxidation of primary alcohols. The π symmetry of the SOMO obtained from the calculations is evident in Figure 6, which depicts the SOMO contoured to 0.55 e/Å3. The significant π covalency of the bonding of the phenoxyl oxygen leads to a distortion of the ring structure with a contraction in bond lengths along the phenoxyl axis relative to the starting (closed-shell) geometry. The figure shows that the highest occupied MO is π antibonding in character, consistent with contraction of the C-O bond distance upon formation of the phenoxyl radical. This contraction, which results from a decrease in antibonding interactions upon loss of one electron, is experimentally observed in the increase of the νC-O,stretch vibrational frequency for the radical compared to the parent phenol.73 The unpaired electron density at each of the atoms in the ring (F) is given in Figure 6E,F. As illustrated in Figure 6A,B, thioether substitution results in a substantial delocalization of spin density onto the sulfur atom with concomitant reductions at the ring carbons and at the phenoxyl oxygen. The calculated g values (Table 2) reflect the trends in the experimental results (Table 1): the cresyl radical shows a dramatic difference in the principal g values which lie in the plane of the phenoxyl ring (g1 and g2), while the o-(methylthio)cresyl radical displays

EPR of Apogalactose Oxidase Radical

J. Phys. Chem., Vol. 100, No. 41, 1996 16745

TABLE 3: Calculated Molecular g Shifts ∆gXX MTC radical all atoms oxygen sulfur cresyl radical all atoms oxygen

∆gYY

∆gXY

0.003 46 0.002 85 0.000 44

0.002 31 0.002 02 0.000 27

-0.000 62 0.001 65 -0.002 14

0.014 72 0.015 61

0.001 03 0.001 09

0.000 08 0.000 00

a near equivalence of these in-plane g values. The orientation of cresyl radical g matrix principal axes in the molecular frame (Figure 6E), determined by the MO calculations, is nearly collinear with the X,Y,Z axis system illustrated in Figure 1. For the MTC radical, however, delocalization of electron spin density onto the sulfur atom results in a rotation of the in-plane principal axes away from X,Y by Γ ) 23.6° (Figure 6F). Table 3 lists the individual atomic contributions to the g shifts from oxygen and sulfur, as well as the total from all atoms, calculated using eqs 3 and 4. It is evident that contributions from the heavy atom substituents (O and S) are the dominant factors which influence the g values in both of these phenoxy species. Discussion The evidence implicating the covalently modified tyrosine Y272 as the radical site in apogalactose oxidase derives from a variety of experiments. X-band EPR spectroscopy of the protein labeled with [β,β-2H]tyrosine indicates that the radical involves a tyrosine residue.8 Comparison of UV absorption spectra of the radical-containing apoenzyme and a simple (methylthio)cresyl model radical supports assignment of the protein radical to the tyrosine-cysteine moiety.47,49 Resonance Raman spectra have identified a perturbed tyrosyl ligand in the radical site of activated galactose oxidase.48 Finally, results of an EPR/ENDOR study of the apogalactose oxidase radical were interpreted as being consistent with a modified tyrosyl radical species.10 The purpose of this study is to characterize the apogalactose oxidase and model radicals through the use of high-frequency EPR spectroscopy and local density functional molecular orbital calculations. High-frequency EPR spectroscopy of these radicals allows precise determination of g values and provides orientational information less readily available from lowfrequency experiments. The contribution of each atom to the molecular g values depends on both the atomic coefficient in the spin-occupied LCAO molecular orbital and the magnitude of the atomic spin-orbit coupling constant. As a result of this dependence, accurate measurement of molecular g values can reveal the effects of atoms with relatively large spin-orbit coupling such as oxygen and sulfur,39,55 whose predominant isotopes are nonmagnetic and therefore exhibit no hyperfine structure. The experimental high-frequency EPR spectra of apogalactose oxidase and the o-(methylthio)cresol radicals are strikingly similar (Figure 3A,C), in notable contrast to the dissimilarity of the X-band spectra (Figure 2A,C). The g values for the two species are nearly identical (Table 1), indicating that the similarities extend to details of electronic structure. The unrestricted LDF-MO calculations performed on the MTC radical model indicate that the odd-alternate spin density distribution of the unsubstituted phenoxyl radical is approximately retained and that a substantial delocalization of spin density onto the sulfur atom of the thioether substituent occurs in the electronic ground state as a result of π covalency; in fact, the sulfur provides the largest single atomic orbital contribution to the SOMO of the MTC radical. Covalent sulfur contributions to the spin-occupied MO of the radical might be expected to

TABLE 4: Oxygen and Sulfur Orbital Contributions to Calculated g Shiftsa

∆gXX (MTC) ∆gXX (cresyl) ∆gYY (MTC) ∆gYY (cresyl)

summation over occupied orbitals (i < j)

summation over unoccupied orbitals (i > j)

total summation

0.005 59 0.015 77 0.005 59 0.001 50

-0.002 13 -0.000 85 -0.003 28 -0.000 46

0.003 46 0.014 92 0.002 30 0.001 04

a Contributions from single center terms involving only oxygen and sulfur (k′ ) O, S in eqs 4).

imply large orbital g shifts corresponding to the relatively large valence shell spin-orbit coupling for that element, as has been observed in EPR studies of other sulfur-containing radicals.74,75 For example, the principal g values for the thiyl radical generated in γ-irradiated single crystals of cysteine hydrochloride are 2.2441, 2.0006, and 1.9837.76 However, although experimentally measured g values for the apogalactose oxidase and the MTC radicals are distinct from those determined for unsubstituted tyrosyl radicals, the influence of the sulfur substituent on the g values is relatively small; the sum of the orbital g shifts observed experimentally in the high-frequency EPR spectra of the S-methyl-substituted phenoxyl radical is essentially equal to that observed for the unsubstituted tyrosyl species (Table 1). The factors responsible for the seemingly small influence of the sulfur on g values can be understood by examination of Table 4, which lists the results of eqs 4 summed over occupied (i < j) and unoccupied (i > j) molecular orbitals including only contributions from the sulfur 3p and/or oxygen 2p atomic orbitals (k′ ) S 3p and/or O 2p). The most striking difference between the MTC and cresyl radicals is in the values of ∆gXX summed over only occupied levels. This difference can be traced to both a smaller O 2p coefficient in the SOMO of MTC (0.435) versus cresyl (0.522) and a larger orbital splitting between the SOMO and the nearest-occupied MO of a′ symmetry for MTC (0.031 hartree) versus cresyl (0.022 hartree). An additional factor evident in Table 4 which leads to both decreased overall g shifts and reduced in-plane g value anisotropy for the MTC radical is a cancellation of terms arising from a sign difference between unoccupied and occupied orbitals in the energy denominators of eqs 3. Two low-lying unoccupied (a′ symmetry) MTC MO’s with energies 0.153 and 0.177 hartree above the SOMO each contain 0.25 S 3p valence character, while the lowest-lying cresyl levels containing significant O 2p character have energies 0.278 and 0.302 hartree above the SOMO. Perturbative mixing of these lower-lying MTC levels leads to larger (negative) contributions to the g shifts, substantially canceling positive contributions from filled orbitals and thus yielding smaller overall g shifts and smaller in-plane g anisotropy compared to the cresyl radical. An interesting consequence of the presence of the sulfur atom in the MTC radical, as predicted by the ab initio calculations, is a rotation of the in-plane g principal axes by 23° relative to those of the cresyl radical. Unfortunately, the near equivalence of g1 and g2 for the MTC and the apogalactose oxidase radicals (Table 1), together with the mainly isotropic proton hyperfine interactions (Vide infra) evidenced in the EPR spectra (Figures 2A,C, 4, and 5), makes experimental verification of this orientation difficult at present. Use of high-frequency, orientation-selective ENDOR spectroscopy to measure the more anisotropic ring proton hyperfine interations (which are too small to be resolved in the EPR spectra) may provide an experimental means to establish the in-plane g axis orientation. Differences between experimentally and theoretically determined g values may be attributed to the fact that the calculations in this work ignore environmental perturbations such as sur-

16746 J. Phys. Chem., Vol. 100, No. 41, 1996

Gerfen et al.

rounding point charges and hydrogen bonds, factors which are known to influence g values of phenoxy radicals.18,41,71 Inclusion of these factors could lead to calculated g values that more closely agree with experimental values. This is particularly relevant for the cresyl calculations, since introduction of perturbations at the phenoxyl oxygen that stabilize the nonbonded electrons and decrease the oxygen pX,Y character of the near-lying molecular orbitals, thus reducing the orbital matrix elements in eq 2a, will be associated with a smaller g shift along the X axis. Work to characterize these effects is underway. Spin densities calculated here for the cresyl radical model, while not agreeing precisely with values determined by ENDOR spectroscopy for the tyrosyl radical of E. coli RDPR,14 reflect the experimentally derived spin density pattern. This fact and the observation that the measured g values (Table 1) follow the trends predicted by the calculations (Table 2) support the overall accuracy of the MO treatment. Thus, the present level of calculation is clearly sufficient to lend insight into both the effect of the thioether substituent on the electron spin density distribution and the manner by which this distribution defines molecular g values and hyperfine coupling constants. Proton hyperfine interactions have been extensively studied in tyrosyl radicals and have yielded information concerning radical spin distributions and the geometry of the exocyclic methylene group.14,18,77 As described above and illustrated in Figure 6A, the unpaired electron spin in an unsubstituted phenoxyl free radical is localized in a molecular orbital of π symmetry with an odd-alternate spin distribution. Significant spin density exists on the phenoxyl oxygen and on the 3, 5 (ortho) and 1 (para) ring carbons, with smaller spin densities elsewhere in the simple radical. This distribution gives rise to moderately strong hyperfine coupling to R ring protons bonded to the ortho carbons and potentially to the β methylene protons.78,79 The isotropic component of β methylene proton hyperfine coupling derives from a hyperconjugation interaction well described by β Aiso ) F(A + B cos2 θ)

(6)

in which F is the spin density on the π conjugate atom adjacent to the methylene group, A and B are constants whose values depend on structural details of the radical, and θ is the angle defined by the methylene C-H bond and the normal to the ring plane.78,80,81 Because this angle varies greatly among samples, low-frequency (9 GHz) EPR spectra of radical species (including the tyrosyl radicals of RDPR,12 photosytem II,17 and prostaglandin synthase15,16) appear qualitatively different in spite of their chemical similarity. The spectra are thus dominated by differences in hyperfine splittings that obscure the fundamental similarities in electronic g shifts that relate directly to intrinsic features of molecular structure. Previous studies of the apogalactose oxidase radical have shown that the X-band EPR spectrum cannot be simulated by assuming strong coupling to three or more protons, as is typically required for (unsubstituted) tyrosyl radicals, but is reproduced by simulations incorporating strong couplings to two protons.10 This result is confirmed by the two resolved hyperfine splittings (0.85 and 1.42 mT) observed at the high-field edge of the 139.5 GHz spectrum (Figure 4). The LDF-MO calculations described here indicate that substantial spin density exists on the ring para carbon (C1), as well as on oxygen and sulfur (Figure 6). However, in contrast to the case for unsubstituted tyrosyl radicals, the calculation predicts relatively small spin density on the ring ortho carbon C5. The correspondingly small hyperfine coupling to H5 predicted from this spin density is inconsistent with either of the splittings observed in Figure 4.82 Moreover, experiments which incorporated ring-deuterated

tyrosine into the protein failed to produce major modifications to the 9 GHz EPR spectrum.10 Thus, neither of the major hyperfine splittings resolved in the EPR spectra of apogalactose oxidase (Figures 2A and 4A) is assigned to the ring proton H5. The incorporation of [β,β-2H]tyrosine into the apoprotein causes the collapse of the larger hyperfine interaction in the X-band EPR spectrum.8 This result and those of the subsequent ENDOR study led to the assignment of this interaction (Ax ) 1.55 mT, Ay ) Az ) 1.42 mT) to the tyrosyl methylene proton.10 In addition, a significant broadening in the [β,β-2H]tyrosine EPR spectrum remains,8 consistent with the presence of an unresolved (∼0.9 mT) hyperfine splitting. The evidence provided by MO calculations predicting significant spin density on the sulfur and small spin density on the ortho carbon C5 (Figure 6F), in conjunction with the observation that this hyperfine interaction remains upon incorporation of both [β,β-2H]- and [ring 2H]tyrosine, leads us to the assignment of this interaction (Ax′ ) 0.95 mT, Ay′ ) 0.80 mT, Az′ ) 0.85 mT) to a cysteine methylene proton (Table 1). The assignment made here of the two major hyperfine couplings in the apogalactose oxidase radical EPR spectrum to β methylene protons on tyrosine and cysteine is distinct from the interpretation given in the previous ENDOR study.10 This earlier study assigned both couplings to tyrosine, one to a β methylene proton bound to C7 and one to the C5 ring proton. ENDOR spectroscopy of the tyrosyl β methylene proton determined its hyperfine principal values to be 43.4 and 39.8 MHz.10 One component of this coupling (1.42 mT) is wellresolved at the high-field edge of the 139.5 GHz echo-detected spectrum (Figure 4). The ENDOR of the second proton revealed a coupling of 0.85 mT, which is also observed at the high-field edge of the 139.5 GHz spectrum. However, the assignment made previously10 of this second coupling to a ring proton (H5) is inconsistent with the MO calculations performed in this study and with previous experiments incorporating selectively deuterated tyrosine in the protein.10 In addition, the hyperfine interaction (1.18, 0.45, 0.85 mT, compatible with a phenyl ring R proton) assigned to the second proton and used to simulate apogalactose oxidase radical EPR spectrum in the ENDOR study10 failed to produce acceptable simulations of the MTC radical spectra obtained at either 9 or 139.5 GHz. This failure casts doubt on the assignment of this coupling to a phenoxy ring R proton, since it is reasonable to expect this coupling to be approximately the same in the model and protein-based radical.83 However, the angular dependence of the hyperfine interaction arising from a β methylene proton could very well produce different couplings in the model and in the protein radicals as a result of differences in side chain orientations in these two samples. We find that the apogalactose oxidase EPR spectra, at both 9 (Figure 2A,B) and 139.5 GHz (Figures 3A,B and 4), can be well-simulated using two protons which both exhibit nearly isotropic coupling (Table 1). The small anisotropy in both interactions is consistent with that expected for β-methylene protons: essentially isotropic hyperfine couplings to methylene protons adjacent to sulfur have been observed in a variety of radicals.55,74,76,84 The MTC EPR spectra (Figures 2C,D and 3C,D) can be reasonably simulated with isotropic couplings to two sets of three equivalent protons (Table 1), consistent with the assignment of six β methylene protons. Substitution of deuterons for protons at C8 of MTC produces spectroscopic results (Figure 5) which verify the assignments made in Table 1 and corroborate the presence of significant spin density on sulfur. Differences in the proton coupling constants between the MTC and apogalactose oxidase can be attributed to differences in side chain orientations. These simulations,

EPR of Apogalactose Oxidase Radical together with the evidence discussed above, lead to the assignment of the apogalactose oxidase hyperfine structure to two β methylene protons, one bound to C7 of tyrosine (with principal values of 1.55, 1.42, 1.42 mT) and the other to C8 of cysteine (0.95, 0.80, 0.85 mT). As an additional check on the sulfur spin density determined from these molecular orbital calculations (and the hyperfine coupling assignment based on this spin density), we can perform a simple calculation to estimate the expected hyperfine coupling for the C8 protons of apogaloctose oxidase and the model compound MTC. In this comparison we assume the validity of eq 6 in describing the hyperfine interaction and neglect the term A since typically A , B.79,84 To estimate the value of B for the C8 proton splitting, a single-crystal study of thiyl radical can serve as a point of departure.76 In this study, a single β proton was observed with an essentially isotropic hyperfine coupling of ∼3.6 mT. Inasmuch as only one C8 proton hyperfine interaction is observed for the apogalactose oxidase as well, we assume the methylene side chain orientation to be similar to that in the single-crystal study. Scaling the spin density localized on sulfur from 1 for the thiyl radical to 0.28 determined by the MO calculations for the apogalacose oxidase model radical yields a C8 proton isotropic hyperfine coupling of ∼1.0 mT as compared to the measured value of 0.87 mT. An analogous calculation for the model compound MTC yields an estimate of 0.67 mT for the three C8 protons85 Versus 0.50 mT used in the simulations (Table 1). The agreement for both the apogalactose oxidase and MTC radicals, while good, must be viewed as tentative in light of the assumptions made. Studies using deuterated tyrosine incorporated into the protein as well as MTC deuterated at C7 will further characterize these hyperfine interactions. The role of the sulfur linkage in the galactose oxidase radical is of interest in the context of the biological function of the radical redox site. The redox potential of the radical-forming tyrosine in galactose oxidase is approximately 410 mV Versus NHE, nearly 500 mV lower than free tyrosine in solution and 260-600 mV lower than the redox-active tyrosines in PSII and in E. coli RDPR. MO calculations of the MTC species indicate a contribution of sulfur in the ground state and the occurrence of low-lying levels of predominantly sulfur character, implying the existence of low-energy excited states involving charge transfer between the phenoxyl π system and the thioether side chain. This feature may account for the appearance of a lowenergy electronic transition in the optical spectrum of o(methylthio)cresyl and the apogalactose oxidase radical, a spectroscopic signature that distinguishes these radicals from the tyrosyl radical found in ribonucleotide reductase. The proximity of Y272, C228, and W290 in galactose oxidase creates the possibility of an extended π network for delocalization of the radical.9 This putative orbital delocalization network has been cited as a possible reason for the unusual stability of the radical in this enzyme compared to tyrosyl radicals generated in solution. However, the similar g values measured for the radicals formed from apogalactose oxidase and the (methylthio)cresol model compound indicate that there is no substantial delocalization of the unpaired spin density into the tryptophan residue. This conclusion agrees with that given in the earlier ENDOR study.10 It is possible that the tryptophan serves to stabilize the radical by sterically hindering the approach of potential reactants, a role similar to that suggested for this motif in another radical containing enzyme, methanol dehydrogenase from Methylobacterium extorquens.86 Conclusions The comparison of g values resolved by high-frequency EPR spectroscopy demonstrates that the o-(methylthio)cresol phe-

J. Phys. Chem., Vol. 100, No. 41, 1996 16747 noxyl radical is an effective model for the tyrosine-cysteine radical of oxidized apogalactose oxidase. The Zeeman interactions for both of these species are approximately axially symmetric, in contrast to those for unsubstituted tyrosyl radicals which have a higher degree of rhombicity. Theoretical determinations of g values using unrestricted LDF-MO calculations predict the observed trends in the experimental data for both the thioether-substituted and unsubstitued tyrosyl radical species. The high-frequency spectra, together with the MO calculations, provide further evidence for the involvement of a covalent cysteine linkage in the apogalactose oxidase tyrosyl radical and are consistent with the radical spin density being localized on the Y272-C228 moiety rather than delocalized throughout an extended π network involving W290. Echo-detected spectroscopy of the apogalactose oxidase radical at 139.5 GHz has revealed proton hyperfine structure which provides orientationally selective information in the Zeeman interaction principal axis system. These resolved hyperfine interactions arise from two protons. Spin density distributions determined from the LDF-MO calculations, together with EPR spectroscopic results on protonated and selectively deuterated o-(methylthio)cresyl radical, are consistent with the assignment of these hyperfine interactions to two β methylene protonssone bound to cysteine and one to tyrosine. The large proton hyperfine couplings in these orientationally disordered (frozen solution) phenoxyl radical systems tend to dominate their EPR spectra at conventional frequencies (9 GHz). The highly variable β methylene proton couplings can make the X-band EPR spectra of chemically similar species appear very different based principally on the orientation of the β methylene side chain. EPR spectra of these species obtained at 139.5 GHz, while still exhibiting resolved hyperfine splittings, are dominated by the Zeeman interaction which may more effectively reflect chemical identity. High-frequency EPR spectroscopy thus provides a means to measure and correlate the Zeeman and hyperfine interactions which, in conjunction with molecular orbital calculations, can provide a detailed characterization of the molecular and electronic structure of the radical species. Acknowledgment. The authors thank Dr. M. M. Whittaker for synthetic and biochemical preparations of the o-(methylthio)cresol and apogalactose oxidase sample and Professor JoAnne Stubbe for providing the ribonucleotide reductase sample. This research was supported by grants from the National Institutes of Health to J.W.W. (GM-46749) and to R.G.G. and D.J.S. (GM-38352 and RR-00995) and by an American Cancer Society postdoctoral fellowship to G.J.G. (PF-3668). References and Notes (1) Avigad, G.; Amaral, D.; Asensio, C.; Horecker, B. L. J. Biol. Chem. 1962, 237, 2736-2743. (2) Kosman, D. J. In Copper Proteins and Copper Enzymes; Lontie, R., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 2, pp 1-26. (3) Whittaker, J. W. In Metal Ions in Biological Systems; Sigel, H., Sigel, A., Eds.; Marcel Dekker: Basel, 1994; pp 315-360. (4) Hamilton, G. A.; Adolf, P. K.; de Jersey, J.; DuBois, G. C.; Dyrkacz, G. R.; Libby, R. D. J. Am. Chem. Soc. 1978, 100, 1899. (5) Whittaker, M. M.; Whittaker, J. W. J. Biol. Chem. 1988, 263, 60746080. (6) Clark, K.; Penner-Hahn, J. E.; Whittaker, M. M.; Whittaker, J. W. J. Am. Chem. Soc. 1990, 112, 6433-6434. (7) Clark, K.; Penner-Hahn, J. E.; Whittaker, M. M.; Whittaker, J. W. Biochemistry 1994, 33, 12553-12557. (8) Whittaker, M. M.; Whittaker, J. W. J. Biol. Chem. 1990, 265, 96109613. (9) Ito, N.; Phillips, S. E. V.; Yadav, K. D. S.; Knowles, P. F. J. Mol. Biol. 1994, 238, 794-814. (10) Babcock, G. T.; El-Deeb, M. K.; Sandusky, P. O.; Whittaker, M. M.; Whittaker, J. W. J. Am. Chem. Soc. 1992, 114, 3727-3734. (11) Prince, R. C. Trends Biochem. Sci. 1988, 13, 286-288.

16748 J. Phys. Chem., Vol. 100, No. 41, 1996 (12) Ehrenberg, A.; Reichard, P. J. Biol. Chem. 1972, 247, 3485-3488. (13) Sahlin, M.; Petersson, L.; Gra¨slund, A.; Ehrenberg, A.; Sjo¨berg, B.-M.; Thelander, L. Biochemistry 1987, 26, 5541. (14) Bender, C. J.; Sahlin, M.; Babcock, G. T.; Barry, B. A.; Chandrashekar, T. K.; Salowe, S. P.; Stubbe, J.; Lindstro¨m, B.; Petersson, L.; Ehrenberg, A.; Sjo¨berg, B.-M. J. Am. Chem. Soc. 1989, 111, 80768083. (15) Karthein, R.; Dietz, R.; Nastainczyk, W.; Ruf, H. H. Eur. J. Biochem. 1988, 171, 313-320. (16) Lassmann, G.; Odenwaller, R.; Curtis, J. F.; DeGray, J. A.; Mason, R. P.; Marnett, L. J.; Eling, T. E. J. Biol. Chem. 1991, 266, 20045-20055. (17) Barry, B. A.; El-Deeb, M. K.; Sandusky, P. O.; Babcock, G. T. J. Biol. Chem. 1990, 265, 20139-20143. (18) Barry, B. A.; Babcock, G. T. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7099. (19) Hoganson, C. W.; Babcock, G. T. Biochemistry 1992, 31, 1187411880. (20) Lane Gilchrist, M., Jr.; Ball, J. A.; Randall, D. W.; Britt, R. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9545-9549. (21) Koulougliotis, D.; Tang, X.-S.; Diner, B. A.; Brudvig, G. W. Biochemistry 1995, 34, 2850-2856. (22) Hori, H.; Yonetani, T. J. Biol. Chem. 1985, 260, 349. (23) Wagner, A. F. V.; Frey, M.; Neugebauer, F. A.; Scha¨fer, W.; Knappe, J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 996-1000. (24) Sivaraja, M.; Goodin, D. B.; Smith, M.; Hoffman, B. M. Science 1989, 245, 738-240. (25) Licht, S.; Gerfen, G. J.; Stubbe, J. Science 1996, 271, 477-481. (26) Parast, C. V.; Wong, K. K.; Kozarich, J. W.; Peisach, J.; Magliozzo, R. S. Biochemistry 1995, 34, 5712-5717. (27) Stubbe, J. Annu. ReV. Biochem. 1989, 58, 257-285. (28) Stubbe, J. Biochemistry 1988, 27, 3893-3900. (29) Whittaker, M. M.; Whittaker, J. W. Biophys. J. 1993, 64, 762772. (30) Branchaud, B. P.; Montague-Smith, M. P.; Kosman, D. J.; McLaren, F. R. J. Am. Chem. Soc. 1993, 115, 798-800. (31) Hoff, A. J., Ed. AdVanced EPR: Applications in Biology and Biochemistry; Elsevier: Amsterdam, 1989. (32) Kevan, L., Bowman, M. K., Eds. Modern Pulsed and Continuous WaVe Electron Spin Resonance; John Wiley & Sons: New York, 1990. (33) Abragam, A.; Bleaney, B. Electron Paramagnetic Resonance of Transition Ions; Dover: New York, 1970. (34) Evelo, R. G.; Hoff, A. J.; Dikanov, S. A.; Tyrshkin, A. M. Chem. Phys. Lett. 1989, 161, 479-484. (35) Fishel, L. A.; Farnum, M. F.; Mauro, J. M.; Miller, M. A.; Kraut, J.; Liu, Y.; Tan, X.-l.; Scholes, C. P. Biochemistry 1991, 30, 19861996. (36) Force, D. A.; Randall, D. W.; Britt, R. D.; Tang, X.-S.; Diner, B. A. J. Am. Chem. Soc. 1995, 117, 12643-12644. (37) Tsvetkov, Y. D. In Proceedings of the International Workshop on Electron Magnetic Resonance of Disordered Systems; Yordanov, N. D., Ed.; World Scientific: Singapore, 1989; pp 15-57. (38) Lebedev, Y. S. In Modern Pulsed and Continuous-WaVe Electron Spin Resonance; Kevan, L., Bowman, M. K., Eds.; John Wiley & Sons: New York, 1990; pp 365-404. (39) Gerfen, G. J.; Bellew, B. F.; Un, S.; Joseph, J.; Bollinger, M.; Stubbe, J.; Griffin, R. G.; Singel, D. J. J. Am. Chem. Soc. 1993, 115, 64206421. (40) Belford, R. L.; Clarkson, R. B. In AdVances in Chemistry Series; Botto, R. E., Sanada, Y., Eds.; American Chemical Society: Washington, DC, 1993; Vol. 229, pp 107-138. (41) Un, S.; Brunel, L.-C.; Brill, T. M.; Zimmermann, J.-L.; Rutherford, A. W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5262-5266. (42) Krinichnyi, V. I. 2-mm WaVe Band EPR Spectroscopy of Condensed Systems; CRC Press: Boca Raton, FL, 1995. (43) Prisner, T. F.; Est, A. v. d.; Bittl, R.; Lubitz, W.; Stehlik, D.; Mo¨bius, K. Chem. Phys. 1995, 194, 361-370. (44) Disselhorst, J. A. J. M.; Meer, H. v. d.; Poluektov, O. G.; Schmidt, J. J. Magn. Reson. A 1995, 115, 183-188. (45) Gulin, V. I.; Dikanov, S. A.; Tsvetkov, Y. D.; Evelo, R. G.; Hoff, A. J. Pure Appl. Chem. 1992, 64, 903-906. (46) Fasanella, E. L.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1969, 62, 299-304. (47) Itoh, S.; Hirano, K.; Furuta, A.; Komatsu, M.; Ohshiro, Y.; Ishida, A.; Takamuku, S.; Kohzuma, T.; Nakamura, N.; Suzuki, S. Chem. Lett. 1993, 2099-2102. (48) Whittaker, M. M.; DeVito, V. L.; Asher, S. A.; Whittaker, J. W. J. Biol. Chem. 1989, 264, 7104-7106. (49) Whittaker, M. M.; Chuang, Y.-Y.; Whittaker, J. W. J. Am. Chem. Soc. 1993, 115, 10029-10035. (50) Barigelletti, F.; Juris, A.; Balzani, V.; Belser, P.; Zewlewsky, A. v. Inorg. Chem. 1983, 22, 3335-3339. (51) Pilgram, K. H.; Medina, D.; Soloway, S. B.; Gaertner, G. U. Method of Production of Hydroxyaryl Thioehters from Sulfonium Compounds. U.S. Patent 3772391, 1973. (52) Salowe, S. P.; Stubbe, J. J. Bacteriol. 1986, 165, 363-366.

Gerfen et al. (53) Becerra, L.; Gerfen, G.; Bellew, B.; Bryant, J.; Hall, D.; Inati, S.; Weber, R. Un, S.; Prisner, T.; McDermott, A.; Fishbein, K.; Kreischer, K.; Temkin, R.; Singel, D.; Griffin, R. J. Magn. Reson., Ser. A 1995, 117, 2840. (54) Prisner, T. F.; Un, S.; Griffin, R. G. Isr. J. Chem. 1992, 32, 357363. (55) van der Donk, W. A.; Stubbe, J.; Gerfen, G. J.; Bellew, B. F.; Griffin, R. G. J. Am. Chem. Soc. 1995, 117, 8908-8916. (56) Hyde, J. S.; Jesmanowiscz, A.; Ratke, J. J.; Antholine, W. E. J. Magn. Reson. 1992, 96, 1-13. (57) Burghaus, O.; Rohrer, M.; Go¨tzinger, T.; Plato, M.; Mo¨bius, K. Meas. Sci. Technol. 1992, 3, 765-774. (58) Ammerlaan, C. A. J.; van der Wiel, A. J. Magn. Reson. 1976, 21, 387-396. (59) Delley, B. J. Chem. Phys. 1990, 92, 508-517. (60) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 12001211. (61) Becke, A. D. J. Chem. Phys. 1988, 88, 2547. (62) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785. (63) Baker, J.; Sheiner, A.; Andzelm, J. Chem. Phys. Lett. 1993, 216, 380-388. (64) Lamin, G. J.; Handy, N. C.; Amos, R. D. Mol. Phys. 1993, 80, 1121-1134. (65) Qin, Y.; Wheeler, R. A. J. Chem. Phys. 1995, 102, 1689-1698. (66) VanVleck, J. H. The Theory of Electric and Magnetic Susceptibilities; Oxford University Press: New York, 1965. (67) Stone, A. J. Mol. Phys. 1963, 6, 509-515. (68) Stone, A. J. Mol. Phys. 1964, 7, 311-316. (69) McCain, D. C.; Hayden, D. W. J. Magn. Reson. 1973, 12, 312330. (70) Prabhananda, B. S.; Felix, C. C.; Hyde, J. S.; Walvekar, A. J. Chem. Phys. 1985, 83, 6121-6127. (71) Burghaus, O.; Plato, M.; Rohrer, M.; Mo¨bius, K.; MacMillan, F.; Lubitz, W. J. Phys. Chem. 1993, 97, 7639-7647. (72) Atkins, P. W.; Symonds, M. C. R. The Structure of Inorganic Radicals; Elsevier: New York, 1967. (73) Mukheerjee, A.; McGlashin, M. L.; Spiro, T. G. J. Chem. Phys. 1995, 99, 4912-4917. (74) Box, H. C.; Budzinski, E. E. J. Chem. Soc., Perkins Trans. 2 1976, 553-555. (75) Preston, K. F.; Sutcliffe, L. H. Magn. Reson. Chem. 1990, 28, 189204. (76) Kou, W. W. H.; Box, H. C. J. Chem. Phys. 1976, 64, 3060-3062. (77) Box, H. C.; Budzinski, E. E.; Freund, H. G. J. Chem. Phys. 1974, 61, 2222-2226. (78) Carrington, A.; McLachlan, A. D. Introduction to Magnetic Resonance; Harper & Row: New York, 1967. (79) Wertz, J. E.; Bolton, J. R. Electron Spin Resonance: Elementary Theory and Practical Applications.; Chapman and Hall: New York, 1986. (80) McLachlan, A. D. Mol. Phys. 1958, 1, 233-240. (81) Heller, C.; McConnell, H. M. J. Chem. Phys. 1960, 32, 15351539. (82) Because H5 is an R proton in a π radical species, its isotropic hyperfine interaction can be modeled by the relation (Aiso ) QF) in which Q is the McConnell constant and F is the spin density on the adjacent π conjugate carbon atom.78,79,81 Assuming a McConnell Q value for the ortho proton similar to that determined experimentally for the tyrosyl radical in E. coli ribonucleotide reductase (|Q| ) 2.48 mT),14 the spin density of 0.08 (Figure 6) on C5 determined by the MO calculation leads to an isotropic hyperfine coupling of approximately 0.2 mT. This is in contrast to the isotropic value of approximately 0.9 mT determined experimentally for the smaller of the two resolved hyperfine interactions. Because the difference in these two values is signficantly greater than that which could arise from uncertainties in the calculated spin densities and in the measured hyperfine coupling constant, we do not assign this hyperfine interaction to the R proton H5. (83) The similarity in g values between the MTC radical and the apogalactose oxidase radical suggests a similarity in electronic structure, and thus equal spin density at C5, for the two species. The chemical similarity of the radicals suggests an equality in McConnell Q values. It thus seems reasonable to expect a similarity in ortho hyperfine interactions between the MTC and apogalactose oxidase radicals. (84) Miura, Y.; Asada, H.; Kinoshita, M.; Ohta, K. J. Phys. Chem. 1983, 87, 3450-3455. (85) For this estimate, we assume that the methyl group in MTC freely rotates at 100 K, and thus cos2 θ (eq 6) exhibits the average value of 0.5 for the spectra displayed in Figure 5.78 We also assume that, in the thiyl radical ENDOR study,76 cos2 θ ∼ 0 for the undetected methylene proton and thus cos2(π/2-2π/3) ∼ 0.75 for the observed proton. Using eq 6 and again scaling the sulfur spin density from 1 to 0.28 yields the estimate of 0.67 mT for the C8 protons of MTC. (86) Avezoux, A.; Goodwin, M. G.; Anthony, C. Biochem. J. 1995, 307, 735-741.

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