G-Tensors of the Flavin Adenine Dinucleotide Radicals in Glucose

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its ... flavins and protein-bound flavin radicals.10-19 To...
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3568

J. Phys. Chem. B 2008, 112, 3568-3574

G-Tensors of the Flavin Adenine Dinucleotide Radicals in Glucose Oxidase: A Comparative Multifrequency Electron Paramagnetic Resonance and Electron-Nuclear Double Resonance Study Asako Okafuji,† Alexander Schnegg,†,‡ Erik Schleicher,† Klaus Mo1 bius,† and Stefan Weber*,†,§ Freie UniVersita¨t Berlin, Fachbereich Physik, Institut fu¨r Experimentalphysik, Arnimallee 14, 14195 Berlin, Germany, Hahn-Meitner-Institut Berlin, Abteilung Silizium PhotoVoltaik Silizium-PhotoVoltaik, Kekule´ strasse 5, 12489 Berlin, Germany, and Albert-Ludwigs-UniVersita¨t Freiburg, Institut fu¨r Physikalische Chemie, Albertstrasse 21, 79104 Freiburg, Germany ReceiVed: September 6, 2007; In Final Form: December 17, 2007

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD•-) and neutral (FADH•) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD•- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX ) 2.00429(3), gY ) 2.00389(3), gZ ) 2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso ) 2.00345(3). The gY-component of FAD•from GO is moderately shifted upon deprotonation of FADH•, rendering the g-tensor of FAD•- slightly more axially symmetric as compared to that of FADH•. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH• radical into the anionic FAD•- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.

Introduction Flavins are the most frequently encountered organic redox cofactors in nature, catalyzing many different reactions of physiological importance.1-3 Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) have the 7,8dimethyl isoalloxazine ring in common but differ in the side chain attached to N(10) (see Figure 1). With their three oxidation states, fully oxidized, one-electron reduced semiquinone, and fully reduced hydroquinone, flavins are involved in one-electron and two-electron-transfer reactions.3 The two radical forms of flavins, the neutral (N(5)-protonated) and the anionic (N(5)deprotonated) semiquinone, play important roles as redox-active intermediates in many enzymatic processes ranging from the biosynthesis and degradation of metabolic compounds4 to bluelight initiated processes such as plant phototropism,5 circadian timing,6 or DNA photorepair.7 In studies of paramagnetic flavin species, application of electron paramagnetic resonance (EPR) has traditionally been very valuable to distinguish between the anionic and neutral radical forms.8,9 Electron-nuclear double resonance (ENDOR) with its increased spectral resolution has provided more detailed information on the molecular structure and the distribution of the unpaired electron spin over the isoalloxazine ring of model * To whom correspondence should be addressed. E-mail: Stefan. [email protected]. † Freie Universita ¨ t Berlin. ‡ Hahn-Meitner-Institut Berlin. § Albert-Ludwigs-Universita ¨ t Freiburg.

Figure 1. Molecular structure and IUPAC numbering scheme of anionic (FAD•-) and neutral (FADH•) flavin radical. R denotes the ribityl adenosine diphosphate side chain.

flavins and protein-bound flavin radicals.10-19 Together with pulsed EPR techniques,20-25 these methods allow, via the detection of hyperfine coupling constants, mapping of the spindensity distribution over the tricyclic heteronuclear aromatic flavin ring system. Such information is a prerequisite for the determination of sites that participate in electron-transfer reactions of flavoenzymes, and is therefore of fundamental importance to unravel the diversity of flavin-mediated biocatalysis. With the recent availability of powerful EPR instrumentation operating at high magnetic fields and high microwave frequencies (up to 14 T/360 GHz in our laboratory26), it is now possible to perform experiments to resolve even the very small g-tensor anisotropy of flavin semiquinones. Previous g-tensor measurements on noncovalently protein-bound neutral flavin radicals revealed that the canonical g-values are very robust against changes in the immediate surroundings of the flavin.27,28 This situation changes when the flavin is covalently attached to the protein, as has been demonstrated in recent experiments on chemically different LOV1 and LOV2 domains of the bluelight receptor phototropin.29 These results suggest that the

10.1021/jp077170j CCC: $40.75 © 2008 American Chemical Society Published on Web 02/27/2008

G-Tensors of FAD Radicals in Glucose Oxidase g-tensor and its anisotropy may be used for an unambiguous identification of the type of flavin radical under consideration. While numerous studies have been carried out on neutral flavin semiquinones, detailed and accurate g-tensor information on flavin anion radicals is still comparatively scarce. A recent multifrequency X-band (9.1 GHz), W-band (94 GHz) EPR, and Q-band (34 GHz) ENDOR study revealed first principal g-values for the anionic flavin semiquinone in Na+-translocating NADH: quinone oxidoreductase (Na+-NQR), but the full anisotropy of the g-tensor could not be resolved with sufficient precision.18 In the present g-tensor study, we focus on the redox-active FAD cofactor of Aspergillus niger (A. niger) glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4 GO) that can be reversibly transformed into its anionic radical form, FAD•-, by decreasing the proton concentration. GO catalyzes the oxidation of β-D-glucose by molecular oxygen to hydrogen peroxide and glucono-δ-lactone, which subsequently hydrolyzes spontaneously to gluconic acid.30 The A. niger protein is composed of two identical subunits with a molecular weight of approximately 80 kDa each.31 Each subunit carries one molecule of noncovalently but, nevertheless, tightly bound coenzyme, FAD, situated about 15 Å below the protein surface.32 GO is an ideal model system for an examination of the two radical forms of the FAD cofactor because the protein can stabilize both the neutral and the anionic semiquinone form depending on the pH, and is quite robust in a wide temperature range.33 The enzyme is also of considerable commercial importance, with the proteins from A. niger and Penicillium amagasakiense being mostly used in glucose sensors.34,35 Finally, a wealth of structural information from X-ray diffraction32,36 and NMR spectroscopy37 is available on the A. niger protein. In this contribution, together with X-band and W-band EPR and pulsed X-band ENDOR, we apply 360-GHz EPR with its significantly increased spectral resolution to determine the full rhombic symmetry of the g-tensor of the FAD anion radical in A. niger GO. Experimental Procedures Sample Preparation. GO from A. niger (Type X-S) was purchased from Sigma-Aldrich and used without further purification. The enzyme was dissolved in a 60 mM phosphate buffer (pH 10 or pH 5) and supplemented with 10 mM EDTA as the electron donor. After the adjustment of the pH, the enzyme solution was deaerated by bubbling with argon gas. For X-band and W-band EPR measurements, the protein preparations were transferred into EPR quartz tubes (3 mm inner diameter for X-band EPR/ENDOR; 0.6 mm inner diameter for W-band EPR) under an argon inert gas atmosphere in the dark. The enzyme samples were then illuminated at 4 °C with white light from a halogen lamp (Streppel Halolux 100 HL, Wermelskirchen-Tente, Germany) to generate GO with the anionic radical form of the flavin at pH 10 or the neutral radical form at pH 5. The samples were then frozen rapidly with liquid nitrogen. For 360-GHz EPR spectroscopy, the enzyme solution was quickly placed onto a sample holder after the illumination and also frozen rapidly with liquid nitrogen. Enzyme concentrations were determined on the basis of the FAD absorbance at 450 nm (450 ) 1.31 × 104 M-1 cm-1).38,39 The redox state of the FAD cofactor was checked systematically by measuring the ground-state absorption spectrum from 300 to 800 nm with a UV/vis spectrophotometer (Varian Cary 100 Scan). EPR Spectroscopy. Continuous-wave (cw) EPR and pulsed EPR/ENDOR spectra at X-band were recorded by using an EPR spectrometer (Bruker Elexsys E580) equipped with a DICEENDOR accessory including a radio frequency (RF) amplifier

J. Phys. Chem. B, Vol. 112, No. 11, 2008 3569 (Amplifier Research 250A250A) and a dielectric-ring ENDOR resonator (Bruker EN4118X-MD-4-W1), which was immersed in a helium gas-flow cryostat (Oxford CF-935). The temperature was regulated to (0.1 K by a temperature controller (Oxford ITC-503S). Magnetic-field-swept electron-spin-echo-detected EPR spectra were recorded with a microwave frequency of 9.735 GHz using a (π/2)-500 ns-π microwave pulse sequence with 64-ns and 128-ns π/2- and π-pulses, respectively. For Daviestype ENDOR spectroscopy, a microwave pulse sequence π-t(π/2)-τ-π with 64-ns and 128-ns π/2- and π-pulses, respectively, and an RF pulse of 10 µs duration starting 1 µs after the first microwave pulse were used. The separation times t and τ between the microwave pulses were selected to be 13 µs and 500 ns, respectively. To avoid saturation effects due to the typically long relaxation times of flavin radicals, the entire pulse pattern was repeated with a frequency of only 200 Hz. All experiments were performed at 80 K. W-band cw EPR spectra were recorded by a commercial EPR spectrometer (Bruker Elexsys E680) using a cylindrical cavity (Bruker Teraflex EN600-1021H), which was immersed in a helium gas-flow cryostat (Oxford) regulated by a temperature controller (Oxford ITC-503). 360-GHz cw EPR experiments were performed with a laboratory-built super-heterodyne 12.8 T/360 GHz quasi-optical spectrometer consisting of a 14-T superconducting magnet (Oxford Teslatron), and a 360-GHz heterodyne microwave board (Farran Technologies and Radiometer Physics).26 Spectra were recorded without a microwave resonator in induction mode with a small amount (about 20 µL) of sample placed onto a sample holder. The probe head was then introduced into the pre-cooled static helium contact cryostat (Oxford Instruments CF1200) inside the superconducting magnet. All experiments were performed at 140 K. Spectral Simulations. For the calibration of the magnetic field in the 360-GHz EPR experiments, the six-line EPR spectrum from a Mn(II)/MgO standard was recorded before and after the measurement of the GO sample. The EPR spectrum of the magnetic-field standard was simulated following the procedure described in the literature.40 The 360-GHz EPR spectrum of GO was simulated using the program “fx-fitx” described in detail elsewhere.27 A nonlinear least-squares fitting algorithm yielded the best agreement between simulated and experimental data for the parameter sets given in Table 1. The fitting routine is based on a trust-region reflective Newton algorithm (Matlab, The Mathworks). In the starting parameter sets of the fitting procedure, the hyperfine couplings of the H(8R) protons, as obtained from pulsed ENDOR, were kept fixed, and only the AZ components of the two nitrogens (I ) 1), 14N(5) and 14N(10), were automatically adjusted together with the canonical values of the g-tensor, gX, gY, and gZ (X, Y, and Z are the principal axes of g), and the orientation-dependent residual line widths ΓX, ΓY, and ΓZ along the respective principal axes of g. Parts of the pulsed X-band ENDOR spectrum were simulated by using the EasySpin toolbox41 (http://www.easyspin.ethz.ch/) to obtain precise values for the hyperfine couplings of the H(8R) and H(5) protons. Error Discussion. The error margins for the canonical g-values strongly depend on the resonance frequency of the spectrometer employed. For W-band EPR data, the optimum accuracy ((0.0001) is determined by the limited resolution of the canonical g-values in the spectrum. Increasing the resonance frequency to 360 GHz may significantly increase the accuracy of the values extracted. In the latter case, the error margins are

3570 J. Phys. Chem. B, Vol. 112, No. 11, 2008

Okafuji et al.

TABLE 1: Magnetic Interaction Parameters of Different Protein-bound Flavin Radicalsa anionic flavin radicals A. niger glucose oxidase pH 10

d

2.00429(3) 2.00389(3) 2.00216(3) 2.00345(3)

Na+-NQRb

neutral flavin radicals A. niger glucose oxidase pH 5

2.00436(2) 2.00402(2) 2.00228(2) 2.00355(2)

2.0043(1) 2.0036(1) 2.0021(1) 2.0035(1)

E. coli DNA photolyasec

gX gY gZ giso

A(H(8R))/MHz

A|| A⊥

(+)11.45(5)e (+)9.95(5)e

A(14N(5))/MHz

A|| A⊥

53(3)f 0(3)f

57.6(5) 2.3(6)

53(5)f 0(5)f

50.1(20)f 0f

51(2)f 0f

52.5(5) 0.2(10)

A(14N(10))/MHz

A|| A⊥

25(3)f 0(3)f

22.8(6) 1.6(6)

30(5)f 0(5)f

31.7(20)f 0f

27(2)f 0f

28.9(6) 2.0(10)

A(H(5))/MHz

Ax Ay Az

-

(+)8.45(5)e (+)6.85(5)e

-

(-)10(5)f (-)33.9(1)e (-)24.1(1)e

(+)8.66(5)e (+)6.80(5)e

(-)8.50(10)e (-)37.00(5)e (-)24.90(5)e

2.00433(5) 2.00368(5) 2.00218(7) 2.00340(6)

Na+-NQRb

g-tensor

(+)11.9 (+)10.0

2.00431(5) 2.00360(5) 2.00217(7) 2.00336(6)

Xenopus laeVis (6-4) photolyased

(+)8.13(5)e (+)6.50(5)e

(-)13.7(2)e (-)38.4(1)e (-)26.1(1)e

2.00425(2) 2.00360(2) 2.00227(2) 2.00337(2) (+)10.0 (+)8.5

(-)0.2(10) (-)38.6(6) (-)25.8(6)

a The signs of hyperfine couplings given in parentheses have not been determined experimentally. b Reference 18. c References 24, 27, and 57. References 23, 28, and 48. e Values obtained from ENDOR spectra. f Values obtained from high-field EPR spectra.

Figure 2. Light-induced changes in the UV/vis absorption spectra of A. niger GO at pH 10 (upper) and pH 5 (lower) in the presence of EDTA recorded at 273 K. The spectra were taken before illumination (solid lines), and after illumination of the sample for the time periods specified.

determined by the error of the isotropic g-value of the Mn(II)/ MgO field standard.40 In both cases, the error margins are typically larger than the confidence intervals obtained from the fitting routine (typically smaller than ( 0.00001 for 360-GHz EPR spectra). Hyperfine couplings may be extracted from pulsed ENDOR spectra with an accuracy of less than (100 kHz. In addition, we have extracted hyperfine parameters from simulations of the high-field EPR spectra where ENDOR data was not available. Because of the inferior spectral resolution of EPR spectra, as compared to ENDOR spectra, the error margins of these values are in the range of several megahertz. Results and Discussion Optical Spectroscopy. Figure 2 shows light-induced spectral changes in the optical absorptions of A. niger GO in the presence of EDTA at pH 10 (upper curves) and pH 5 (lower curves). UV/vis spectra at 273 K were collected from the dark-adapted system (solid line) and immediately after different periods of

white-light illumination. From the literature it is well-known that, by photoreduction of GO, two different protonation states of the FAD semiquinonesthe anionic FAD•- radical and the neutral FADH• radicalscan be generated depending on the pH. These may be distinguished on the basis of their characteristic UV/vis spectra.33 For the pH-10 solution, the intensity of the absorption band around 450 nm, which originates from the fully oxidized FAD, decreases as a function of illumination time. Concomitantly, two absorption bandssone sharp peak located at 400 nm, and a second broader band at 500 nm with a shoulder extending to 550 nmsgrow upon illumination. These absorption bands are characteristic for FAD•-.33 At pH 5, however, the generation of FADH• can be deduced from the broad absorption around 570 nm.33 Our results are in good agreement with those from previous reports on GO and allow for a clear discrimination between the two radical species. It should be noted, however, that the radical concentration generated by photoreduction of GO at pH 5 is significantly smaller than that obtained at pH 10. The EPR and ENDOR spectra on the pH-5 sample presented in the subsequent section, therefore, show inferior signal-tonoise ratios as compared to the pH-10 sample. Pulsed X-band EPR and ENDOR Spectroscopy. The presence or absence of certain proton hyperfine couplings, such as the one arising from H(5), provides evidence (in addition to the UV/vis data) on whether the neutral or the anionic flavin radical form is present in a flavoprotein. In general, such a differentiation on the basis of the EPR line width alone is often not possible, because anionic and neutral flavin radicals frequently show quite similar EPR spectra. This is also the case for the flavin semiquinone in GO, where the FAD•- radical present at pH 10 exhibits only a marginally smaller EPR line width as compared to FADH• at pH 5 (see insets of Figure 3). Therefore, pulsed (Davies) X-band proton ENDOR spectra from GO at pH 10 (upper) and pH 5 (lower) in the frequency range between 4 and 40 MHz have been recorded at a magnetic field B0 ) 346.45 mT, corresponding to g ) 2.0034 (see arrows in the insets of Figure 3). Compared to published ENDOR spectra on anionic flavin radicals that were recorded in the cw mode,42 the pulsed ENDOR data presented here show increased spectral resolution and significantly enhanced sensitivity. This is valid in particular for the detection of very anisotropic and, hence, broad hyperfine signals such as the one from H(5), which had previously not been observed (see lower spectra in Figure 3).

G-Tensors of FAD Radicals in Glucose Oxidase

Figure 3. Pulsed X-band EPR (insets) and (Davies) ENDOR spectra from A. niger GO recorded at 80 K at pH 10 (upper curves) and pH 5 (lower curves). ENDOR spectra in an RF range between 4 and 40 MHz were subsequently recorded with the same microwave frequency at the magnetic field positions indicated with an arrow in the EPR spectra (see insets). Assigned proton hyperfine couplings are marked.

In the weak-coupling limit (hyperfine coupling , nuclear Zeeman coupling), a pair of ENDOR lines is detected per group of magnetically equivalent protons, separated by the orientationdependent (anisotropic) hyperfine coupling constant, A. To first order, each line pair, ν( ) |νH ( (A/2)|, is symmetrically placed around the proton Larmor frequency, νH ) gHβHB0/h () 14.72 MHz for a magnetic field of B0 ) 346.45 mT in X-band ENDOR). βH and gH are the Bohr magneton and nuclear g-factor, respectively, and h is the Planck constant. For experiments under solid-state conditions with non-oriented frozen protein solutions, the hyperfine lines are inhomogeneously broadened as a result of the superposition of signals originating from all possible orientations of flavin molecules with respect to the direction of the external magnetic field. The principal values of the hyperfine coupling tensors are then extracted from the peak and inflection points of the powder patterns of the ENDOR signals. The central so-called matrix-ENDOR signal extends from about 13 to 16.5 MHz and includes hyperfine couplings from protons only weakly interacting with the unpaired electron spin, which is delocalized over the aromatic π-plane of the isoalloxazine moiety. Such small hyperfine couplings (|A| e 1.25 MHz) originate from protons in the protein backbone within the cofactor binding pocket, from protons of water molecules surrounding the flavin, and also from weakly coupled protons directly attached to the 7,8-dimethyl isoalloxazine ring, namely, H(3), H(7R) and H(9).43,44 The overlapping splittings contributing to the central parts of the ENDOR spectra cannot be unambiguously assigned solely on the basis of presently available ENDOR data. For a full assignment, high-field ENDOR experiments45,46 providing better orientation selection and thereby even higher spectral resolution on proteins containing specifically isotope-labeled FAD are required. Such experiments are presently projected in our laboratory; the results will be presented

J. Phys. Chem. B, Vol. 112, No. 11, 2008 3571 elsewhere. In the following we will only discuss those features of the ENDOR spectra that can be unambiguously assigned by comparison with previously published experimental16,18 and theoretical43,44 data on anionic and neutral flavin radicals. Prominent spectral features are observed at 11 and 18.5 MHz, and at 9.5 and 20 MHz for the protein solutions adjusted to pH 5 and pH 10, respectively. The tensorial line shapes are of axial symmetry and arise from the hyperfine couplings of the β-protons of the methyl group attached to C(8). Typically, methyl groups rotate freely about their C-C bond, even at very low temperatures. Hence, if this rotation is fast on the ENDOR time scale, one averaged hyperfine tensor for all three protons of the methyl group is observed. Signals of the H(8R) hyperfine tensor are, in general, easily detected by proton-ENDOR on flavins, and are considered to be sensitive probes of the electronspin density on the outer xylene ring of the isoalloxazine moiety.17,23 By spectral simulations, the principal values of the H(8R) hyperfine tensors were determined: A|| ) 11.45 MHz and A⊥ ) 9.95 MHz for the anionic FAD•- radical at pH 10, and A|| ) 8.45 MHz and A⊥ ) 6.85 MHz for the neutral FADH• radical at pH 5, yielding isotropic hyperfine couplings of 10.45 and 7.4 MHz, respectively. As has been shown in a number of experimental10,11 and theoretical43 studies on flavin radicals, the H(8R) hyperfine values of FAD•- are, in general, considerably larger than those of FADH•. This is because deprotonation of N(5) results in a significant redistribution of the unpaired electron spin from the pyrazine and pyrimidine rings of the isoalloxazine moiety toward the less polar xylene ring. In addition to the H(8R) hyperfine couplings, we were able to assign the ENDOR signals from hyperfine splittings of 8.2 MHz (pH 10) and 5.2 MHz (pH 5) as originating from the H(6) proton. The assignment is aided by predictions of hyperfine couplings using density functional theory.43 The hyperfine coupling of H(6) follows the trend observed for H(8R): it increases when going from the neutral radical to the anion radical, thus supporting the build-up of spin density on the outer xylene ring upon N(5) deprotonation. The 11.3-MHz splitting in the spectrum recorded at pH 5 arises from one or both H(1′) protons attached to the carbon C(1′) of the ribityl side chain next to N(10). The isotropic hyperfine coupling of this β-proton is determined by the spin density at the neighboring N(10) atom in the π-plane of the isoalloxazine ring. According to the McConnell relation,47 A(Hβ) ) Fπ(N(10)) (B′+ B′′ cos2 θ) depends on the spatial orientation of the ribityl side chain. From the two empirical parameters, B′ and B′′, B′ is typically very small, i.e., B′ ≈ 0. θ is the dihedral angle between the plane normal of the π-system and the projected C(1′)-H(1′) bond. The large coupling observed in FADH• of GO implies a small angle θ, which is indeed observed in the high-resolution three-dimensional structure obtained from X-ray crystallography.36 That this splitting is not clearly visible in the anion radical spectra at pH 10 might be due to the reduced spin density at N(10) in FAD•- (as compared to FADH•), resulting in a considerably smaller H(1′) coupling and, therefore, spectral overlap with the H(8R) and H(6) resonances. On the other hand, also a minor spatial rearrangement of the ribityl side chain resulting in a modified angle θ cannot be excluded. At low pH, a broad resonance is observed in the region between 27 and 33 MHz. Its counterpart on the low-frequency side is not clearly discernible because parts of the resonances are folded back at 0 MHz and, furthermore, are overlapping with signals from the nitrogens (centered around 1 MHz) in the isoalloxazine ring in FAD. This broad signal is assigned to H(5) in FADH•, which has been shown previously for the neutral

3572 J. Phys. Chem. B, Vol. 112, No. 11, 2008 radical in DNA photolyase to have a very large and anisotropic hyperfine coupling constant.24 As expected for the anion radical, this resonance is missing for GO at pH 10. This is, thus, a second proof for the presence of pure anionic and neutral radical states at the respective pH values. X-band, W-band, and 360-GHz cw EPR Spectroscopy. In Figure 4 the X-band, W-band, and 360-GHz cw EPR spectra of the FAD cofactor in its stable anionic form bound to GO at pH 10 are shown. From a qualitative inspection of the data it is apparent that the spectral shape significantly changes when increasing the resonant magnetic field by a factor of 40. The EPR signals are strongly inhomogeneously broadened by unresolved nitrogen and proton hyperfine couplings. This leads to a single unresolved EPR line at X-band frequencies and corresponding magnetic fields, where the Zeeman broadening from the anisotropy ∆g of g is small. At W-band frequencies, the line exhibits some asymmetry, due to partly resolved g-tensor anisotropy. Furthermore, a modulation of the EPR intensity originating from nitrogen hyperfine couplings becomes observable in the high-field part of the spectrum (see arrows in the trace W-band of Figure 4). At 360 GHz, the g-tensor anisotropy becomes fully resolved and the principal values gX, gY, and gZ can be read off the spectrum (Figure 4). By combining results from a multi-frequency X-band and W-band EPR study with Q-band ENDOR data, Barquera et al. extracted the principal g-values for an anionic flavin radical in Na+-NQR (see Table 1).18 These authors revealed the g-tensor components by Simplex fittings to the second derivative of the experimental EPR spectra. This procedure relies on the assumption that the applied fitting algorithm finds a unique set of magnetic interaction parameters representing the coupling situation of the electron spin. However, in cases where g and the hyperfine interactions are not fully resolved in the EPR spectrum, which for flavin radicals is the case at W-band frequencies, the adjustable parameters remain strongly correlated. This may lead to ambiguities that cannot be disentangled solely on the basis of numerical procedures. An unambiguous determination of the principal g-values can only be obtained by performing EPR measurements at even higher resonance frequencies and magnetic fields, for which the high-field condition is fulfilled, i.e., the inhomogeneous broadening due to unresolved hyperfine interactions is small compared to that from the anisotropy of g. Under this condition, the fully resolved g-tensor pattern is yielded. This condition is met by our 360GHz high-field EPR experiments. The well-resolved spectrum is characteristic for a randomly oriented radical with a g-tensor of rhombic symmetry (gX * gY * gZ). Because of the enhanced orientation selection and the dramatic increase of g-tensor resolution, the AZ components of the hyperfine tensors of 14N(5) and 14N(10) can now be directly extracted from the splitting of the gZ component (see arrows in trace 360 GHz of Figure 4). Further support that the spectrum results from the anionic flavin radical and not from the neutral radical is obtained from the absence of a splitting of the gY component. Such a splitting is typically observed for neutral flavin radicals and is due to the Y-component of the hyperfine tensor of H(5).27,28 Using the principal values of the hyperfine tensor of H(5) obtained from pulsed X-band ENDOR,24 we have shown recently that information on the orientation of the principal axes of g with respect to the molecular coordinate frame of a neutral flavin radical can be extracted.27,48 Because H(5) is absent in anionic flavin radicals, such an analysis of the orientation of the g-tensor could not, of course, be performed for FAD•-. Hence, an unambiguous determination of the g-tensor orientation

Okafuji et al.

Figure 4. X-band (upper), W-band (middle), and 360-GHz (lower) cw EPR spectra (first derivative) of the FAD cofactor of GO in its stable anionic radical form, FAD•-, at pH 10. Experimental conditions: X-band: microwave frequency, 9.731 GHz; microwave power, 0.632 mW; modulation amplitude, 0.1 mT (modulation frequency, 100 kHz); temperature, 80 K. W-band: microwave frequency, 93.890 GHz; microwave power, 3.2 × 10-8 W; modulation amplitude, 0.3 mT (modulation frequency, 100 kHz); temperature, 80 K. 360-GHz: microwave frequency, 360.04 GHz; microwave power, 0.2 mW; modulation amplitude, 0.4 mT (modulation frequency, 7 kHz); temperature, 140 K. Note that the 360-GHz EPR spectrum was detected in induction mode without a microwave resonator. Calculated W-band and 360-GHz spectra are shown as dashed lines. The simulations have been obtained by least-squares fittings to the experimental data using the g and hyperfine values given in Table 1. Inhomogeneous line width parameters (Γ) for the W-band EPR simulation were ΓX ) 1.3 mT, ΓY ) 1.0 mT, and ΓZ ) 0.8 mT, and those for 360-GHz were ΓX ) 1.4 mT, ΓY ) 1.0 mT, and ΓZ ) 1.0 mT.

in an anionic flavin radical would require more time-consuming single-crystal EPR studies performed at high microwave frequencies and magnetic fields. Figure 4 also shows the results of spectral simulations of W-band and 360-GHz spectra obtained by using identical

G-Tensors of FAD Radicals in Glucose Oxidase

Figure 5. Calculated (dashed line) and experimental (solid line) W-Band EPR spectra (first derivative) of the neutral flavin radical cofactor bound to GO. For better comparability, the W-band EPR spectrum of the anionic flavin radical is also shown as a dotted line. Experimental conditions: microwave frequency, 93.890 GHz; microwave power, 3.2 × 10-8 W; modulation amplitude, 0.3 mT (modulation frequency, 100 kHz); temperature, 80 K. Inhomogeneous line width parameters (Γ) for the EPR simulation were ΓX ) 1.3 mT, ΓY ) 1.2 mT, and ΓZ ) 1.2 mT.

parameter sets (see Table 1). The parameters of the anionic flavin radical of GO, gX ) 2.00429(3), gY ) 2.00389(3), gZ ) 2.00216(3), and the AZ hyperfine components of N(5) and N(10), were extracted from least-squares fittings of spectral simulations to the experimental 360-GHz EPR spectra. Figure 5 shows that the experimental W-band cw EPR spectrum together with spectral simulations of the FAD cofactor in its stable neutral radical form (FADH•) bound to GO at pH 5. 360-GHz EPR spectra of FADH• could not be obtained, because the neutral radical reoxidizes quickly at low pH under aerobic conditions to yield the fully oxidized FADοx. [The presence of oxygen cannot be rigorously avoided during the insertion of the sample into the spectrometer.] The radical concentration is thus too low for the presently available detection sensitivity of our instrumentation. Spectral simulations of the W-band cw EPR spectrum yielded the g-principal components gX ) 2.0043(1), gY ) 2.0036(1), and gZ ) 2.0022(1). The AZ components of the N(5) and N(10) hyperfine couplings could not be resolved. As is typical for g-tensor data obtained from W-band EPR data, the increased error margins reflect the limited spectral resolution due to unresolved hyperfine interactions. In Table 1, EPR parameters extracted from fits of numerical simulations to the experimental 360-GHz EPR spectrum of the anionic flavin radical bound to GO are given together with results from Barquera et al. on the anion radical in Na+-NQR,18 and the respective values of neutral flavin radicals bound to different flavoproteins.27-29 Moreover, unambiguously assigned resonances from ENDOR spectra are also listed. As is obvious from Table 1, the canonical g-values of neutral flavin radicals noncovalently bound in different proteins are very robust against changes in the immediate protein surroundings of the flavin cofactor. We observe that, within the experimental error, the gX- and gZ-components for an anionic flavin radical, FAD•-, are almost identical with the respective values from FADH•, despite the fact that the electronic distribution in the frontier orbitals of the two protonation forms of flavin semiquinones should be quite different, as has been confirmed by X-band

J. Phys. Chem. B, Vol. 112, No. 11, 2008 3573 ENDOR (see above). Apparently, both gX and gZ are insensitive against local changes in the electron-spin density, but react only on global changes of the chemical structure of a flavin. On the other hand, the gY-component of FAD•- from GO responds moderately on the deprotonation of FADH•, rendering the g-tensor of FAD•- slightly more axially symmetric as compared to the one of FADH•. We note that, in high-field EPR studies of nitroxide radicals,49 quinones,50,51 and tyrosines,52 usually the gX principal component is more responsive toward changes in the polarity and/or hydrogen-bonding situation than gY, whereas, in this study on a flavin, only gY shows a noticeable shift. This difference presumably lies in the different chemical structure and, hence, different symmetries of flavins and nitroxides or para-quinones. The latter have a well-defined symmetry axis with X aligned along the N-O or CdO bonds, respectively, and the electron-spin densities on the oxygens are high. Hence, they are particularly sensitive to changes of hydrogen bonding in this direction. Flavins, on the other hand, have lower symmetry with the two carbonyl groups being meta-positioned. Furthermore, the spin density in flavins is mostly localized on C(4a) and N(5) rather than on the carbonyl groups. Taking together these observations, it is reasonable that, in neutral and anionic flavin semiquinones, the gY principal value probes more sensitively changes in the chemical structure and modulations of local spin densities than gX and gZ. The difference between the gX and gY components, the asymmetry parameter gX - gY, is approximately 0.0007 for all neutral flavin radicals, whereas it becomes significantly smaller for anionic flavin radicals, i.e., gX - gY ≈ 0.0004. This is most likely due to the fact that, in flavin anion radicals, the unpaired electron spin is more uniformly delocalized over the entire 7,8-dimethyl isoalloxazine moiety, thus resulting in a more symmetric g-tensor, whereas, in the neutral radical, the spin density is more asymmetrically localized on the central pyrazine and the outer pyrimidine rings. This notion is corroborated by the hyperfine coupling constants of the H(8R) and H(6) protons obtained by ENDOR. Their values are, in general, larger in the flavin anion radicals, thus implying larger spin density on the xylene ring as compared to the neutral radicals. Since the quantity gX - gY is conserved among the different flavoproteins examined so far by high-field EPR, we suggest that gX - gY can be used as a probe to distinguish between the anionic and neutral flavin radicals in proteins. To summarize, we have examined the anionic flavin radical of GO by EPR performed at 360-GHz, W-band, and X-band as well as by pulsed X-band ENDOR. Because of the high spectral resolution of the frozen-solution cw EPR at 360 GHz, the anisotropy of the g-tensor of the anionic flavin radical could be fully resolved for the first time. Furthermore, the AZ components of hyperfine couplings from N(5) and N(10) could be determined with high accuracy, benefiting from the excellent orientation selection. These values are compared with those obtained from EPR on the neutral flavin radical of GO. We anticipate that, in future work, high-field EPR in conjunction with ENDOR spectroscopy will play an important role in identifying the protonation state of flavin radicals and their overall binding situation to the protein surroundings (covalently versus noncovalently bound). This will apply especially in cases where spectrophotometry does not allow for an unambiguous characterization of these parameters because of the pronounced variability in the optical absorption properties of flavin radicals in general. Whereas, in some cases, the optical absorption spectra of similar flavin radicals show band shifts of up to 100 nm, the principal values of the g-tensor are very

3574 J. Phys. Chem. B, Vol. 112, No. 11, 2008 robust and, thus, are ideally suited as a measure of the overall electronic situation. Furthermore, in studies of flavoprotein samples at intermediate pH values where mixtures of neutral and anion radicals are expected, pulsed ENDOR spectroscopy correctly returns the relative ratio of the flavin protonation states. For example, in measurements of GO samples at pH 6, the ENDOR spectrum showed a double-peak feature that originated from the different H(8R) hyperfine couplings of FADH• and FAD•- (data not shown), whereas the UV/vis spectrum exhibits only contributions from FADH•. The GO reaction is supposed to proceed through a “pingpong” mechanism.53 The catalytic cycle is divided into one reductive and one oxidative half-reaction. While the reductive half-reaction, a net hydride transfer from the anomeric C-H bond of glucose to FAD,39,53 is largely pH independent, the oxidative half-reaction, which results in the oxidation of FADHby oxygen (forming hydrogen peroxide as a product), strongly depends on the pH.54,55 Although site-directed mutagenesis studies have implied a major role of a conserved histidine residue (His-516 in A. niger GO) in the latter reaction,55,56 the exact mechanism is still not fully understood. Given its pronounced pH dependence, we assume that the different electronic spin-density distributions of FAD•- and FADH•, in particular at the C(4a) position, may have an impact on the efficiency of the enzyme’s redox reactivity. Clearly, future highfield EPR and ENDOR studies are aimed at a characterization of the flavin’s reactive positions in terms of their unpaired spin densities, and how they are modulated by the interaction of the cofactor with the protein. The results of such experiments will enable us to deepen our understanding of flavin-mediated biocatalysis in general and the functioning of GO in particular. Acknowledgment. We thank Professor Robert Bittl (Freie Universita¨t Berlin) for the use of EPR instrumentation and continuous support. This work was supported by the Deutsche Forschungsgemeinschaft (Sfb-498 and SPP-1051). References and Notes (1) Massey, V. Biochem. Soc. Trans. 2000, 28, 283-296. (2) Fraaije, M. W.; Mattevi, A. Trends Biochem. Sci. 2000, 25, 126132. (3) Joosten, V.; van Berkel, W. J. H. Curr. Opin. Chem. Biol. 2007, 11, 195-202. (4) Massey, V. FASEB J. 1995, 9, 473-475. (5) Briggs, W. R.; Christie, J. M. Trends Plant Sci. 2002, 7, 204210. (6) Berndt, A.; Kottke, T.; Breitkreuz, H.; Dvorsky, R.; Hennig, S.; Alexander, M.; Wolf, E. J. Biol. Chem. 2007, 282, 13011-13021. (7) Weber, S. Biochim. Biophys. Acta 2005, 1707, 1-23. (8) Edmondson, D. E. Biochem. Soc. Trans. 1985, 13, 593-600. (9) Kay, C. W. M.; Weber, S. In Electron Paramagnetic Resonance; Gilbert, B. C., Davies, M. J., Murphy, D. M., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2002; Vol. 18, pp 222-253. (10) Kurreck, H.; Bock, M.; Bretz, N.; Elsner, M.; Kraus, H.; Lubitz, W.; Mu¨ller, F.; Geissler, J.; Kroneck, P. M. H. J. Am. Chem. Soc. 1984, 106, 737-746. (11) Kurreck, H.; Bretz, N. H.; Helle, N.; Henzel, N.; Weilbacher, E. J. Chem. Soc., Faraday Trans. 1 1988, 84, 3293-3306. (12) Medina, M.; Vrielink, A.; Cammack, R. Eur. J. Biochem. 1994, 222, 941-947. (13) Medina, M.; Gomez-Moreno, C.; Cammack, R. Eur. J. Biochem. 1995, 227, 529-536. (14) DeRose, V. J.; Woo, J. C. G.; Hawe, W. P.; Hoffman, B. M.; Silverman, R. B.; Yelekci, K. Biochemistry 1996, 35, 11085-11091. (15) Fan, C.; Teixeira, M.; Moura, J.; Moura, I.; Huynh, B.-H.; Gall, J. L.; Peck, H. D., Jr.; Hoffman, B. M. J. Am. Chem. Soc. 1991, 113, 20-24. (16) Kay, C. W. M.; Feicht, R.; Schulz, K.; Sadewater, P.; Sancar, A.; Bacher, A.; Mo¨bius, K.; Richter, G.; Weber, S. Biochemistry 1999, 38, 16740-16748.

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