Multifrequency Pulsed EPR Studies of Biologically Relevant Manganese(II) Complexes.

Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H(2)O)(6)](2+), Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echo-detected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented.

31P NMR probes of chemical dynamics: paramagnetic relaxation enhancement of the (1)H and (31)P NMR resonances of methyl phosphite and methylethyl phosphate anions by selected metal complexes.

Methyl phosphite ((CH(3)O)P(H)(O)(2)(-); MeOPH) and methylethyl phosphate ((CH(3)O)P(OCH(2)CH(3))(O)(2)(-); MEP) are two members of a class of anionic ligands whose (31)P T(2) relaxation rates are remarkably sensitive to paramagnetic metal ions. The temperature dependence of the (31)P NMR line broadenings caused by the Mn(H(2)O)(6)(2+) ion and a water-soluble manganese(III) porphyrin (Mn(III)TMPyP(5+)) indicates that the extent of paramagnetic relaxation enhancement is a measure of the rate at which the anionic probes come into physical contact with the paramagnetic center (i.e., enter the inner coordination shell); that is, piDeltanu(par) = k(assn)[M], where Deltanu(par) is the difference between the line widths of the resonance in paramagnetic and diamagnetic solutions, and k(assn) is the second-order rate constant for association of the phosphorus ligand with the metal, M. Comparison of the (31)P T(1) and T(2) relaxation enhancements shows that rapid T(2) relaxation by the metal ion is caused by scalar interaction with the electronic spin. Relaxation of the phosphorus-bound proton of MeOPH ((1)H-P) by Mn(III)TMPyP(5+) displayed intermediate exchange kinetics over much of the observable temperature range. The field strength dependence of (1)H-P T(2) enhancement and the independence of the (31)P T(2) support these assertions. As in the case of the (31)P T(2), the (1)H-P T(2) relaxation enhancement results from scalar interaction with the electronic spin. The scalar coupling interpretation of the NMR data is supported by a pulsed EPR study of the interactions of Mn(H(2)O)(6)(2+) with the P-deuterated analogue of methyl phosphite, CH(3)OP((2)H)(O)(2)(-). The electron to (31)P and (2)H nuclear scalar coupling constants were found to be 4.6 and 0.10 MHz, respectively. In contrast, the effects of paramagnetic ions on the methoxy and ethoxy (1)H resonances of MeOPH and MEP are weak, and the evidence suggests that relaxation of these nuclei occurs by a dipolar mechanism. The wide variation in the relaxation sensitivities of the (1)H and (31)P nuclei of MeOPH and MEP permits us to study how differences in the strengths of the interactions between an observed nucleus and a paramagnetic center affect NMR T(2) relaxations. We propose that these anion ligand probes may be used to study ligand-exchange reactivities of manganese complexes without requiring variable temperature studies. The (31)P T(2) is determined by chemical association kinetics when the following condition is met: (T(2M,P)/T(2M,H))(Deltanu(P)/Deltanu(HP) - 1) < 0.2 where T(2M,P) and T(2M,H) are the transverse relaxation times of the (31)P and (1)H nuclei when the probe is bound to the metal, and Deltanu(P) and Deltanu(HP) are the paramagnetic line broadenings of the (31)P and (1)H-P nuclei, respectively. We assert that the ratio T(2M,P)/T(2M,H) can be estimated for a general metal complex using the results of EPR and NMR experiments.

Dual-mode EPR study of Mn(III) salen and the Mn(III) salen-catalyzed epoxidation of cis-beta-methylstyrene.

Dual-mode electron paramagnetic resonance (EPR), in which an oscillating magnetic field is alternately applied parallel or perpendicular to the static magnetic field, is a valuable technique for studying both half-integer and integer electron spin systems and is particularly useful for studying transition metals involved in redox chemistry. We have applied this technique to the characterization of the Mn(III) salen (salen = N,N'-ethylene bis(salicylideneaminato)) complex [(R,R)-(-)-N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminomanganese(III)], with an S = 2 integer electron spin system. Furthermore, we have used dual-mode EPR to study the Mn salen complex during the Mn(III) salen-catalyzed epoxidation of cis-beta-methylstyrene. Our study shows that the additives N-methylmorpholine N-oxide (NMO) and 4-phenylpyridine-N-oxide (4-PPNO), which are used to improve epoxidation yields and enantioselection, bind to the Mn(III) center prior to the epoxidation reaction, as evidenced by the alteration of the Mn(III) parallel mode EPR signal. With these additives as ligands, the axial zero-field splitting values and (55)Mn hyperfine splitting of the parallel mode signal are indicative of an axially elongated octahedral geometry about the Mn(III) center. Since the dual-mode EPR technique allows the observation of both integer and half-integer electron spin systems, Mn oxidation states of II, III, IV, and potentially V can be observed in the same sample as well as any radical intermediates or Mn(III,IV) dinuclear clusters formed during the Mn(III) salen-catalyzed epoxidation reaction. Indeed, our study revealed the formation of a Mn(III,IV) dinuclear cluster in direct correlation with expoxide formation. In addition to showing the possible reaction intermediates, dual-mode EPR offers insight into the mechanism of catalyst degradation and formation of unwanted byproducts. The dual-mode technique may therefore prove valuable for elucidating the mechanism of Mn(III) salen catalyzed reactions and ultimately for designing optimum catalytic conditions (solvents, oxidants, and additives such as NMO or 4-PPNO).

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study.

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster.

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).

Pulsed and parallel-polarization EPR characterization of the photosystem II oxygen-evolving complex.

Photosystem II uses visible light to drive the oxidation of water, resulting in bioactivated electrons and protons, with the production of molecular oxygen as a byproduct. This water-splitting reaction is carried out by a manganese cluster/tyrosine radial ensemble, the oxygen -evolving complex. Although conventional continuous-wave, perpendicular -polarization electron paramagnetic resonance (EPR) spectroscopy has significantly advanced our knowledge of the structure and function of the oxygen-evolving complex, significant additional information can be obtained with the application of additional EPR methodologies. Specifically, parallel-polarization EPR spectroscopy can be use to obtain highly resolved EPR spectra of integer spin Mn species, and pulsed EPR spectroscopy with electron spin echo-based sequences, such as electron spin echo envelope modulation and electron spin echo-electron nuclear double resonance, can be used to measure weak interactions obscured in continuous-wave spectroscopy by inhomogeneous broadening.

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II.

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II.

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.

Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH.

Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704-708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine-manganese coupling show that the histidine ligation is present in PS II centers showing the S(2) multiline EPR signal in the pH-range 4.2-9.5.

An electron spin-echo envelope modulation (ESEEM) study of the QA binding pocket of PS II reaction centers from spinach and Synechocystis.

We have used electron spin-echo envelope modulation spectroscopy (ESEEM) to characterize the protein-cofactor interactions present in the QA- binding pocket of PS II centers isolated from spinach and Synechocystis. We conclude that the ESEEM spectrum of QA- is the result of interactions of the S = 1/2 electron spin of QA- with the I = 1 nuclear spins of the peptide nitrogens of two different amino acids. One peptide nitrogen has ESEEM peaks near 0.7, 2.0, 2.85, and 5.0 MHz with isotropic and dipolar hyperfine couplings of Aiso = 2.0 MHz and Adip = 0.25 MHz, respectively. On the basis of these hyperfine couplings we predict the existence of a strong hydrogen bond between QA- and the peptide nitrogen with a hydrogen bond distance of about 2 A. We have not identified the amino acid origin of this peptide nitrogen. By using amino acid specific isotopic labeling in conjunction with site-directed mutagenesis, we demonstrate that the second peptide nitrogen is that of D2-Ala260, with ESEEM peaks near 0.6 and 1.5 MHz and an isotropic hyperfine coupling, Aiso, less than 0.2 MHz. This small isotropic coupling suggests that the D2-Ala260 peptide nitrogen at best forms a weak hydrogen bond with QA-.

Electronic Structure of the Aqueous Vanadyl Ion Probed by 9 and 94 GHz EPR and Pulsed ENDOR Spectroscopies and Density Functional Theory Calculations.

The aqueous vanadyl ion ([VO(H(2)O)(5)](2+)) has been investigated by X-band EPR, 94 GHz W-band EPR, and ESE-ENDOR. These experiments reveal information about the hyperfine (|A(xx)| = 208.5 MHz, |A(yy)| = 208.5 MHz, |A(zz)| = 547.0 MHz), and nuclear quadrupole coupling (|e(2)qQ| = 5.6 MHz) of the (51)V nucleus. The measured nuclear quadrupole coupling parameters are compared to values determined by density functional theory calculations (|e(2)qQ| = 5.2 MHz). These theoretical calculations illustrate that axial ligands and molecular distortions can alter the magnitude of the nuclear quadrupole interaction.

The 23 and 17 kDa extrinsic proteins of photosystem II modulate the magnetic properties of the S1-state manganese cluster.

An S1-state parallel polarization "multiline" EPR signal arising from the oxygen-evolving complex has been detected in spinach (PSII) membrane and core preparations depleted of the 23 and 17 kDa extrinsic polypeptides, but retaining the 33 kDa extrinsic protein. This S1-state multiline signal, with an effective g value of 12 and at least 18 hyperfine lines, has previously been detected only in PSII preparations from the cyanobacterium sp. Synechocystis sp. PCC6803 [Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J., and Britt, R. D. (1998) J. Am. Chem. Soc. 120, 447-448]. It is absent in PSII spinach membrane and core preparations that either fully retain or completely lack the 33, 23, and 17 kDa extrinsic proteins. The S1-state multiline signal detected in spinach PSII cores and membranes has the same effective g value and hyperfine spacing as the signal detected in Synechocystis PSII particles. This signal provides direct evidence for the influence of the extrinsic PSII proteins on the magnetic properties of the Mn cluster.

Hydrogen bonding, solvent exchange, and coupled proton and electron transfer in the oxidation and reduction of redox-active tyrosine Y(Z) in Mn-depleted core complexes of photosystem II.

The redox-active tyrosines, Y(Z) and Y(D), of Photosystem II are oxidized by P680+ to the neutral tyrosyl radical. This oxidation thus involves the transfer of the phenolic proton as well as an electron. It has recently been proposed that tyrosine Y(Z) might replace the lost proton by abstraction of a hydrogen atom or a proton from a water molecule bound to the manganese cluster, thereby increasing the driving force for water oxidation. To compare and contrast with the intact system, we examine here, in a simplified Mn-depleted PSII core complex, isolated from a site-directed mutant of Synechocystis PCC 6803 lacking Y(D), the role of proton transfer in the oxidation and reduction of Y(Z). We show how the oxidation and reduction rates for Y(Z), the deuterium isotope effect on these rates, and the Y(Z)* - Y(Z) difference spectra all depend on pH (from 5.5 to 9.5). This simplified system allows examination of electron-transfer processes over a broader range of pH than is possible with the intact system and with more tractable rates. The kinetic isotope effect for the oxidation of P680+ by Y(Z) is maximal at pH 7.0 (3.64). It decreases to lower pH as charge recombination, which shows no deuterium isotope, starts to become competitive with Y(Z) oxidation. To higher pH, Y(Z) becomes increasingly deprotonated to form the tyrosinate, the oxidation of which at pH 9.5 becomes extremely rapid (1260 ms(-1)) and no longer limited by proton transfer. These observations point to a mechanism for the oxidation of Y(Z) in which the tyrosinate is the species from which the electron occurs even at lower pH. The kinetics of oxidation of Y(Z) show elements of rate limitation by both proton and electron transfer, with the former dominating at low pH and the latter at high pH. The proton-transfer limitation of Y(Z) oxidation at low pH is best explained by a gated mechanism in which Y(Z) and the acceptor of the phenolic proton need to form an electron/proton-transfer competent complex in competition with other hydrogen-bonding interactions that each have with neighboring residues. In contrast, the reduction of Y(Z)* appears not to be limited by proton transfer between pH 5.5 and 9.5. We also compare, in Mn-depleted Synechocystis PSII core complexes, Y(Z) and Y(D) with respect to solvent accessibility by detection of the deuterium isotope effect for Y(Z) oxidation and by 2H ESEEM measurement of hydrogen-bond exchange. Upon incubation of H2O-prepared PSII core complexes in D2O, the phenolic proton of Y(Z) is exchanged for a deuterium in less than 2 min as opposed to a t(1/2) of about 9 h for Y(D). In addition, we show that Y(D)* is coordinated by two hydrogen bonds. Y(Z)* shows more disordered hydrogen bonding, reflecting inhomogeneity at the site. With 2H ESEEM modulation comparable to that of Y(D)*, Y(Z)* would appear to be coordinated by two hydrogen bonds in a significant fraction of the centers.

Proximity of acetate, manganese, and exchangeable deuterons to tyrosine YZ. in acetate-inhibited photosystem II membranes: implications for the direct involvement of YZ. in water-splitting.

The environment of the photosystem II YZ. radical, trapped in the "split-signal" form, is examined in acetate-treated PSII membranes using pulsed EPR methods. The split-signal line shape is simulated with dipolar and exchange couplings to the Mn cluster of 1260 and -28 MHz, respectively. The 1260-MHz dipolar coupling corresponds to a Mn-YZ. distance of 3.5 A in the point dipole limit. A 0.117-MHz dipolar coupling is observed between nonexchangeable deuterons of methyl-deuterated acetate and YZ.. This interaction is modeled with a 3.1-A distance between an acetate methyl group deuteron and the phenoxy oxygen of YZ*. Since acetate inhibition is competitive with Cl-, this result strongly suggests a close proximity between YZ. and the Cl- cofactor binding site. Analysis of pulsed ENDOR and ESEEM experiments investigating the proximity of deuterons exchanged into the vicinity of YZ. after incubation in 2H2O-enriched buffer demonstrates that YZ. trapped in the split-signal form participates in two hydrogen-bonding interactions, in contrast to YD*, which forms a single hydrogen bond. This result is inconsistent with a simple electron transfer role for YZ* and provides direct experimental evidence for a role for YZ* in proton or hydrogen atom transfer.

Proximity of the manganese cluster of photosystem II to the redox-active tyrosine YZ.

Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine YZ. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines YZ and YD. The distance between the YZ tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine YZ, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.

Temperature-dependent pulsed electron paramagnetic resonance studies of the S2 state multiline signal of the photosynthetic oxygen-evolving complex.

The electron spin-lattice relaxation rate (1/T1) of the g = 2 "multiline" manganese electron paramagnetic resonance (EPR) signal arising from the photosystem II oxygen-evolving complex poised in the S2 state has been directly measured over the temperature range of 4.2-11 K via the inversion-recovery pulsed EPR technique. The electron spin echo amplitude of the g = 2 "multiline" signal varies inversely with temperature over this range, indicating a ground spin state Curie law behavior in agreement with our previously reported work [Britt et al. (1992) Biochim. Biophys. Acta 1140, 95-101]. Results of a plot of the natural log of the electron spin-lattice relaxation rate versus reciprocal temperature are consistent with an Orbach mechanism serving as the dominant relaxation pathway for the "multiline" signal in this temperature range. The slope of the plot indicates that an excited spin state manifold exists 36.5 cm-1 above the ground-state manifold that gives rise to the "multiline" signal.

Electron spin echo envelope modulation spectroscopy of the molybdenum center of xanthine oxidase.

The pulsed EPR technique of electron spin echo envelope modulation (ESEEM) has been utilized to examined both the 'very rapid' and 'desulfo inhibited' Mo(V) signals of xanthine oxidase in order to probe for magnetic interactions with nitrogen, phosphorus and hydrogen nuclei. No 14N modulation is observed in the 'desulfo inhibited' EPR signal, indicating that histidine is unlikely to be a ligand to molybdenum. Strong 14N modulation is observed in the 'very rapid' EPR signal formed with 2-hydroxy-6-methylpurine substrate bound to molybdenum. We interpret this modulation as arising from nitrogens of the bound purine substrate. This interpretation is consistent with the present evidence indicating that the purine ring present in the species giving rise to the 'very rapid' EPR signal is coordinated to the molybdenum center through the catalytically introduced hydroxyl group. No modulation is observed from non-exchangeable deuterons in experiments performed with deuterated 2-hydroxy-6-methylpurine. Given the signal-to-noise level of the spectra, the lack of modulation indicates that each of the substrate methyl group deuterons is greater than 4.9 A from the Mo(V). The deuteron removed from the C8 position in the binding of the substrate is also exchanged to a site or sites greater than 4.9 A from the Mo(V) in the time-course of sample preparation. Moderately deep deuteron modulation arises from exchangeable sites. A large portion of this modulation can be accounted for by the exchangeable N7 deuteron of the 2-hydroxy-6-methylpurine substrate, which we estimate to be approximately 3.2 A from the molybdenum. Additional exchangeable deuterons on the protein or within the buffer must be present within 5 A of the molybdenum to account for the remaining modulation. No modulation from weakly-coupled 31P nuclei is observed in either the 'desulfo inhibited' or 'very rapid' EPR signal.