JBIC (1999) 49 :292–301 Q SBIC 1999

ORIGINAL ARTICLE

Roman M. Davydov 7 Joanne SmiejaSergei A. Dikanov 7 Yan Zang 7 Lawrence Que, Jr.Michael K. Bowman

EPR properties of mixed-valent m-oxo and m-hydroxo dinuclear ironcomplexes produced by radiolytic reduction at 77 K

Received: 27 June 1998 / Accepted: 25 February 1999

R. M. Davydov1 7 J. Smieja 7 S. A. Dikanov2

M. K. Bowman (Y)Macromolecular Structure and Dynamics, WR WileyEnvironmental Molecular Science Laboratory, Pacific NorthwestNational Laboratory, Richland, WA 99352-0999, USAe-mail: michael.bowman6pnl.gov, Tel: c1-509-3763299Fax: c1-509-3762303

R. M. DavydovInstitute of Chemical Physics, Russian Academy of SciencesMoscow 117977, Russia

J. SmiejaDepartment of Chemistry, Gonzaga University, SpokaneWA 99258, USA

S. A. DikanovInstitute of Chemical Kinetics and Combustion, RussianAcademy of Sciences, Novosibirsk 630090, Russia

Y. Zang 7 L. Que, Jr.Department of Chemistry, University of MinnesotaMinneapolis, MN 55455, USA

Present addresses:1R. M. Davydov, Department of Chemistry, NorthwesternUniversity, Evanston, IL 60208, USA2S. A. Dikanov, IERC, University of Illinois, Urbana, IL 61801USA

Abstract Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in froz-en solutions forms kinetically stabilized, mixed-valentspecies in high yields that model the mixed-valent sitesof non-heme, diiron proteins. The mixed-valent speciestrapped at 77 K retain ligation geometry similar to theinitial diferric clusters. The shapes of the mixed-valentEPR signals depend strongly on the bridging ligands.Spectra of the Fe(II)OFe(III) species reveal an Sp1/2ground state with small g-anisotropy as characterizedby the uniaxial component (gz–gav /2~0.03) observableat temperatures as high as F100 K. In contrast, hy-droxo-bridged mixed-valent species are characterizedby large g-anisotropy (gz–gav /210.03) and are observa-ble only below 30 K. Annealing at higher temperaturescauses structural relaxation and changes in the EPR

characteristics. EPR spectral properties allow the oxo-and hydroxo-bridged, mixed-valent diiron centers to bedistinguished from each other and can help character-ize the structure of mixed-valent centers in proteins.

Key words Mixed-valent species 7 Dinuclear iron 7EPR 7 Radiolytic reduction

Abbreviations Hr: Hemerythrin 7 R2: R2 subunit ofclass 1 ribonucleotide reductase 7 MMO: Methanemonooxygenase 7 PAP: Purple acid phosphatase 7Uf: Uteroferrin 7 Rb: Rubrerythrin 7 metHr:Methemerythrin

Introduction

Non-heme dinuclear iron-oxo centers are a commonstructural component in the active sites of a number ofproteins and enzymes with diverse functions [1, 2, 3, 4,5]. These centers have important functional roles in he-merythrin (Hr) [3]; the R2 subunit of class 1 ribonu-cleotide reductase (R2) [6, 7, 8]; methane monooxygen-ase (MMO) [9, 10, 11, 12]; purple acid phosphatase(PAP) [13], including uteroferrin (Uf) [1, 2, 4]; stearoylACP desaturase [14]; rubrerythrin (Rb) [15]; and ferri-tin [16, 17]. The bridging ligand is an oxo ligand in thediferric R2 subunits [6, 7, 8], methemerythrin (metHr)[3], Rb [15], and desaturase [14], and a hydroxo ligandin MMO [12], PAP [13], and Uf [1, 2, 4].

In general, the dinuclear iron sites can exist in threeoxidation states: diferric Fe(III)-Fe(III), mixed-valentFe(II)-Fe(III), and fully reduced Fe(II)-Fe(II) [1, 2, 3,4, 18]. The ligand structure and electronic properties ofthe diferric clusters have been characterized by a varie-ty of physical methods. Progress has been aided by ex-tensive studies of synthetic m-oxo and m-hydroxo difer-ric complexes that model the structural and electronicproperties of the diferric sites [1, 2, 18, 19].

In contrast, fewer spectroscopic studies have beenperformed [1, 2, 3, 4] on the diiron centers in the

293

Scheme 1

mixed-valent state. Mixed-valent synthetic complexeshave been reported but most possess a phenolato oxy-gen atom as the bridge [1, 20, 21, 22, 23, 24, 25, 26, 27],with only a few possessing an oxo or hydroxo bridge,e.g., [{Fe(acacen)2}(m-O)(m-Na)]2 [28], 4a [29], and 4d[30] (refer to Scheme 1 for an explanation of the label-ing of the compounds used and Scheme 2 for the li-gands). In addition, a few oxo-bridged complexes havebeen reported with metastable mixed-valent states: 1ain dimethylformamide [31], 2a [32], 3a in acetonitrile[33], and [{[(tris-pic-Me-en)(Cl)Fe]2(m-O)}(Cl)(OH)][34].

Chemical reduction of the diferric state is generallyan unproductive route for generating mixed-valent der-ivatives of synthetic dinuclear iron complexes becauseof their instability. However, the diferric sites of Es-cherichia coli R2, metHr, MMO hydroxylase, and a fewmodel complexes were recently reported to be singlyreduced efficiently in frozen solution at 77 K utilizingmobile electrons generated by g-irradiation, with yieldsof 30–70% [9, 35, 36, 37, 38]. Because molecular motionis limited at 77 K, the mixed-valent clusters produced

by cryogenic reduction are trapped in a constrainednon-equilibrium state [35, 36, 39] with coordination,particularly by the bridging ligands, similar to that ofthe initial diferric state. In the case of the model com-pounds examined here, we focus primarily on the factthat the bridging ligands do not protonate or deproton-ate at 77 K immediately after cryogenic reduction. Suchchanges are observed only after annealing to highertemperatures. In the context of dinuclear iron centersin proteins, the enzymatic cycle sometimes involveschanges in the number of ligands or their geometric ar-rangement around the metals. This method of cryogen-ic reduction allows preparation of an EPR-active statewithout any change in the position of the ligands. Thisis confirmed in the present work, where we see no evi-dence of changes in ligation at 77 K after irradiation. Itis also expected from the results on the irradiation offrozen solutions of Agc [40], where the structure andeven the ligand geometry remained unchanged at 77 Kafter reduction by an electron.

The mixed-valent clusters can be examined withEPR spectroscopy whereas the initial diferric clusters

294

Scheme 2

are EPR silent due to antiferromagnetic coupling be-tween the iron centers. The metHr and R2 mixed-val-ent states generated at 77 K have axial EPR signalswith low g-anisotropy observable without noticeablebroadening up to 100–110 K. This differs dramaticallyfrom their respective mixed-valent forms prepared bystandard chemical reduction at room temperature,where the coordination of the iron changes [35, 36]. If acorrelation between EPR parameters and structure canbe made for model compounds, then EPR studies ofthe mixed-valent state of cryogenically reduced pro-teins can be used to probe the structure of the proteindiferric state. Such a correlation has been suggested onthe basis of a limited range of model compounds [37].

In this paper, we demonstrate that mixed-valentstates of a broad range of synthetic m-oxo- and m-hy-droxo-bridged diiron complexes can be generated bylow-temperature, radiolytic reduction. Further, we de-

monstrate that the EPR signals are characteristic of thestructure of the complex and closely resemble those re-sulting from the low-temperature, one-electron reduc-tion of metHr, R2, and MMO hydroxylase. We haveexamined structural factors that determine the proper-ties of the Sp1/2 EPR signals for a variety of thesecomplexes.

Materials and methods

All reagents and solvents were purchased from commercialsources and used as received unless otherwise noted.

Synthesis of complexes

The dinuclear iron(III,III) complexes were prepared according topublished procedures: 1a [41]; 1b [42]; 2b [43]; 2c [44]; 2d [45]; 3a[33]; 3b [46]; 3c [47]; 4b and 4c [48].

295

Sample preparation

The complexes were dissolved in acetone/acetonitrile to form1–4 mM solutions except for 1a which was prepared in 2-methyl-tetrahydrofuran or dimethylformamide. The solutions were froz-en in nominally 4 mm diameter quartz tubes (Wilmad Glass) andtypically formed a glass. The samples were irradiated by 60Co g-rays while immersed in liquid nitrogen at a nominal dose rate of1.2 Mrad/h for 3–5 h. Irradiated samples were annealed in a cold,regulated nitrogen gas stream followed by recooling to 77 K.

EPR measurements

EPR spectra were recorded on a Bruker ESP 380E X-band EPRspectrometer using 100 kHz field modulation with an Oxford In-strument ESR-910 liquid helium cryostat. A strong EPR signalfrom radiation-induced free radicals partially overlaps signals ofthe mixed-valent species. The irradiated samples were typicallyannealed at 114 K for 40–50 s before measurement, thereby de-creasing the radical signals by a factor of 30–100 while reducingthe mixed-valent EPR signals by less than a factor of 2. Compari-son of doubly integrated spectra with a Cu(II) standard indicatedthat, for typical diferric compounds, saturating yields of themixed-valent state were within 40–60% of the starting material.Thus, even after annealing to remove the majority of free radi-cals, millimolar concentrations of mixed-valent states are presentfor spectroscopic measurements.

Results and discussion

The complexes studied were chosen to fall into severalcategories based on the type of Fe-X-Fe core. Theseare shown in Scheme 1. Scheme 2 shows the relevantligands. Scheme 1 shows the general structure of thecompounds but is not intended to specify the valencestate or charge of the complexes. In general, the com-plexes are synthesized in the diferric state with antifer-romagnetically coupled Fe(III) (Sp5/2), which has noEPR signal. One-electron reduction produces the EPR-active, mixed-valent form with the same or similarstructure. The valence state of a particular complex un-der discussion will be evident in context. The mono-bridged complexes 1a and 1b contain two iron ionscoordinated to a common oxo ion. In the symmetrical1a, four nitrogens of a porphyrin ligand coordinateeach iron. In the unsymmetrical 1b, one iron atom iscoordinated with a pentadentate nitrogenous ligandand the oxo bridge, forming a distorted octahedron,while the other iron atom is coordinated to three chloroions and the oxo bridge in a tetrahedral fashion withC3v local symmetry [42].

The dibridged complexes, 2b, 2c, and 2d, differmainly in the additional bridging ligand (Ypacetateanion, OH–, or OH–777H2O). The irons in each com-plex are coordinated by m-O2–, Y, and four nitrogenatoms of L in a distorted octahedral geometry [43, 44,45]. In the tribridged complexes with the general struc-ture 3 or 4, two six-coordinate iron atoms are bridgedby a m-oxo or m-hydroxo ligand and two bidentate car-boxyl or phosphate groups (Y) and capped by faciallycoordinating tridentate nitrogen ligands. Each iron issurrounded by a distorted octahedral array of ligands[33, 46, 47, 48].

Fig. 1 EPR spectra of mixed-valent forms of 1b (a), 2b (b), 2c (c),and 2d (d) in acetone/acetonitrile glass reduced radiolytically at77 K, 100 kHz field modulation amplitude 0.5 mT, gain 5000, 1mW microwave power at 9.46 GHz, temperature 77 K for a, b, dand 40 K for c. A pair of sharp lines centered at gp2.00 and splitby about 50.5 mT in most spectra arise from trapped hydrogenatoms in the sample or the sample tube

Mixed-valent oxo-bridged dinuclear iron complexes

Figure 1a shows the EPR spectrum of monobridged 1bin acetone/acetonitrile irradiated at 77 K. In addition tosignals at gp2.0 from trapped free radicals created byradiolysis, an axial signal with effective gkp1.960,gIIp1.93, and gav~2.0 is clearly observed. This axialpattern is not observed in mononuclear complexes ofiron(III) with the same ligands, i.e., [(N5)Fe(OEt)]2c

and FeCl3, in acetone/acetonitrile irradiated at 77 K.The shape of the gav~2 signal is independent of tem-perature from 5 to 110 K. It resembles spectra ofmixed-valent species with high-spin, antiferromagneti-cally coupled irons from 2a and 3a produced by chemi-cal reduction as well as from the cryogenically reducedR2 and metHr. The reported and newly measured ef-fective g-tensors of mixed-valent species in this series ofcomplexes are tabulated in Tables 1 and 2, respectively.The symmetric monobridged diferric complex 1a (notshown) also gives a mixed-valent species with an axialEPR spectrum (gkp1.97, gIIp1.935) similar to that inFig. 1a. We assign these species to the mixed-valent dii-ron species produced when radiolytically generatedmobile electrons reduce the diferric complex.

The shape of the EPR signal for cryogenically re-duced, mixed-valent derivatives of dibridged iron-oxocomplexes depends on the additional bridging ligand.Mixed-valent 2b has an unresolved signal (Fig. 1b) withg;1.96, nearly identical to that from chemical reduc-tion [32]. Compound 2d yields a species, observable toat least 100 K, with an axial EPR signal (Fig. 1d,gkp1.96, gIIp1.93), while 2c has a much larger g-aniso-tropy (Fig. 1c, gkp1.95, gIIp1.85) and has an EPR sig-

296

Table 1 Principal values of the effective g-tensor for mixed-valent forms of oxo dinuclear iron centers in proteins and model com-pounds

Compound Preparationa g-tensor gav Ref.

1a E 1.937, 1.96, 1.96 1.95 31[{Fe(TPA)}2Cl2(O)] C 1.92, 1.96, 1.96 1.947 32[{Fe(phen)2(H2O)}2(ClO4)4(O)] R 1.96, 1.96, 1.91 1.94 37

1.98, 1.96, 1.91 1.95[{Fe(phen)2Cl2}2(ClO4)4(O)] R 1.98, 1.96,1.91 1.95 37[{Fe(bipy)2(H2O)}2(ClO4)4(O)] R 1.96, 1.96,1.91 1.94 37[{Fe(4,4b-Me2bipy)2(H2O)}2(ClO4)4(O)] R 1.96, 1.96,1.91 1.94 37[{Fe(EDTA)}2Na4(O)] R 1.95, 1.95, 1.89 1.93 37[{Fe(pyH)Cl3}2(O)] R 1.95, 1.95, 1.95 1.95 372a YpSO4

2– C 1.95 322a YpPO4

3– C 1.95 322a YpMoO4

2– C 1.92, 1.95, 1.95 1.94 322b C 1.95, 1.95, 1.95 1.95 32[{Fe(phen)2}2(ClO4)3(O)(OAc)] R 1.98, 1.95, 1.91 1.95 37[{Fe(bipy)2}2(ClO4)3(O)(OAc)] R 1.98, 1.95, 1.91 1.95 37[{Fe(dipic)(H2O)}2(OH)2] R 1.94, 1.78, 1.63 1.78 37[{Fe(pic)}2(OH)2] R 1.94, 1.88, 1.80 1.87 373a C 1.89, 1.95, 1.95 1.93 33[{Fe(bipy)(H2O)}2(ClO4)2(O)(OAc)2] R 1.98, 1.95, 1.95 1.96 37

1.98, 1.95, 1.93 1.95[{Fe(bipy)(H2O)}2(ClO4)2(O)(MPDP)] R 1.96, 1.94, 1.94 1.94 37

1.96, 1.94, 1.91 1.941.96, 1.94, 1.92 1.94

E. coli R2 protein R 1.818, 1.936, 1.936 1.897 35Methemerythrin R 1.853, 1.954, 1.954 1.92 35Methemerythrin azide R 1.923, 1.944, 1.944 1.94 35Mouse R2 protein C 1.601, 1.73, 1.92 1.75 57, 58HSV R2 protein C 1.63, 1.75, 1.93 1.77 57, 58Semi hemerythrinR C 1.65, 1.86, 1.94 1.81 49Semi hemerythrin azide C 1.50, 1.82, 1.90 1.81 49Rubrerythrin C 1.57, 1.76, 1.98 1.77 59Methane mono-oxygenase (M. trichosporium) R 1.79, 1.86, 1.94 1.87 9Methane mono-oxygenase (M. trichosporium) C 1.77, 1.86, 1.95 1.86 60

1.75, 1.86, 1.95Methane mono-oxygenase (M. capsulatus) C or R 1.71, 1.86, 1.94 1.83 9Fe(II)-Fe(III) ferritin C 1.77, 1.88, 1.95 1.83 16, 17PAP at pHp3.1 C 1.65, 1.78, 1.94 1.79 61Uteroferrin C 1.63, 1.78, 1.95 1.79 62[Fe2(BPMP)2(O2CCHMe)2] C 1.06, 1.61, 1.61 1.43 21[Fe2(bimp)(O2CCHMe)2] C 1.72, 1.81, 1.97 1.83 20[Fe2(bimp)(O2CMe)2] C 1.79, 1.79, 1.98 1.85 20[Fe2(DBE)2(O2CPh)2] C 1.79, 1.79, 1.94 1.84 22[Fe2(HXTA)(O2CMe)2] C broad 234a C 1.56, 1.56, 1.96 1.69 29

a E, electrochemical; C, chemical; R, radiolytic

Table 2 Principal values of the effective g-tensor for mixed-valent forms produced by radiolytic reduction at 77 K from m-oxo- andm-hydroxo-bridged diiron(III) complexes

Complex Before annealing After annealing

g-tensor gav g-tensor gav

1a 1.935, 1.97, 1.97 1.961b 1.934, 1.96, 1.96 1.95 1.932, 1.96, 1.96 1.952b 1.956 1.956 1.9562c 1.846, 1.95, 1.95 1.915 1.89, 1.95, 1.95 1.932d 1.935, 1.96, 1.96 1.95 1.93, 1.96, 1.96 1.953a 1.91, 1.931, 1.954 1.93 1.87, 1.93, 1.93 1.91

1.57, 1.57, F2.0 F1.713b 1.95, 1.946, 1.946 1.95 1.60, 1.70, F1.98 F1.763c 1.925 1.925 1.63, 1.70, 1.99 1.774b 1.64, 1.74, F2.0 F1.79 1.60, 1.695, 1.99 1.764c 1.64, 1.72, F2.0 F1.79 1.63, 1.69, 1.99 1.77

297

Fig. 2 EPR spectra of mixed-valent forms of 3a (a), 3b (b), and3c (c) in acetone/acetonitrile glass reduced radiolytically at 77 K,100 kHz field modulation amplitude 0.5 mT, gain 5000, 1 mW mi-crowave power at 9.46 GHz, temperature 77 K for a, b, and c. Thefree radical signal is omitted for clarity

Fig. 3 EPR spectra of mixed-valent forms of 4b (a) and 4c (c) inacetone/acetonitrile glass reduced radiolytically at 77 K beforeannealing and (b) and (d) after annealing for 3 min at 115 K, re-spectively. 100 kHz field modulation amplitude 1.0 mT, gain 5000,1 mW microwave power at 9.46 GHz, temperature 7 K

nal that broadens above 65 K. Unfortunately, simulta-neous variation in the structure of the tetradentate li-gand, L, in 2b-d makes it difficult to determine the ef-fect of the bridging ligand, Y, on the spectral propertiesof the mixed-valent forms.

The effect of the bridge and polydentate ligands ismore clearly observed in the triply bridged diiron com-plexes 3a-c. The mixed-valent form of 3c has an unre-solved EPR signal at X-band frequencies (Fig. 2c,gp1.925). Replacement of the phosphate bridge in 3cby an acetate, to make 3b, introduces a slight anisotro-py (Fig. 2b, gkp1.946 and gIIp1.95). In 3a the rhombicEPR signal has a fairly small spread in g-values (Fig. 2a,g1p1.95, g2p1.93, and g3p1.91).

Mixed-valent hydroxo-bridged dinuclear ironcomplexes

Cryogenic reduction of hydroxo-bridged diferric com-plexes 4b and 4c produces highly anisotropic EPR sig-nals (Fig. 3), observable only below 25 K. Before an-nealing, 4c has a rhombic lineshape (Fig. 3c) withg1F2.0, g2p1.72, and g3p1.64. The low-field feature isburied under the strong free radical spectrum and be-comes observable after annealing at 114 K for 90 s(Fig. 3d). (The small, narrower feature at gp1.925 isdue to the mixed-valent species derived from 3c presentas a contaminant.) These signals are similar to those re-ported for 4a, the only crystallographically character-ized mixed-valent diiron complex with a hydroxobridge [29].

The stark contrast in the g-anisotropy and relaxationbehavior of the EPR signals of the mixed-valent diiron-(II,III) species derived from oxo- and hydroxo-bridged

diiron(III) complexes strongly suggests that the proton-ation state of the bridging oxygen atom affects theseEPR properties. These differences can be understoodby postulating that the bridge determines the strengthof the antiferromagnetic interaction [37] as it does inthe diferric complexes. The best method for measuringJ is based on the magnetic susceptibility of the sample.It has been applied to two other mixed-valent com-plexes [20, 29] but required data collected over the tem-perature range 5–300 K. The samples studied here con-tain not only the mixed-valent species, but also radia-tion-generated free radicals and the diferric precursors– at least three different paramagnetic species. In addi-tion, the free radicals, and in some cases the mixed-val-ent species, are unstable and decay or change above110 K. This drastically restricts the range of tempera-tures at which measurements can be made, precludingany quantitative analysis of susceptibility data. A sec-ond method for measuring J is based on the relaxationof the EPR signal from the mixed-valent species [37]. Ifsimple models in which an Orbach-Aminov processdominated the spin-lattice relaxation described the re-laxation, then an estimate of J could be obtained fromthe temperature dependence of the spin-lattice relaxa-tion rate. We applied both continuous-wave (CW) andpulsed methods for measuring relaxation to a few ofthe mixed-valent species. Although the CW measure-ments produced normal looking saturation curves, thepulsed measurements revealed that the spin-lattice re-laxation was strongly non-exponential, raising funda-mental questions concerning the interpretation of theCW saturation curves. When we attempted, neverthe-less, to continue processing the CW data and the slow-est exponential component in the pulsed measure-

298

ments, different temperature dependences were ob-tained. In addition, the temperature range over whichthese measurements could be made was restricted byboth the stability of the mixed-valent species and theirrapid relaxation above 30 K. Consequently, the result-ing temperature dependences fit an Orbach-Aminovprocess no better than a Raman process or a mixture ofcross relaxation and spin-lattice relaxation. Becausethese practical problems create fundamental difficultiesin obtaining accurate values for J, the readily accessibleEPR parameters became the focus for probing thestructure of the mixed-valent complexes.

The J values of the diiron(III) precursors are tabu-lated [1] and they correlate well with the EPR proper-ties observed, indicating that the nature of the bridgingligand exerts similar control over the exchange cou-pling in both the diferric and the mixed-valent states.The readily accessible EPR parameters are thus a prac-tical substitute for the more fundamental but inaccessi-ble J values of the mixed-valent states. Because of thevery simple spin Hamiltonian used to derive the valuesof J in the diferric complexes, their utility was citedmostly “for comparative purposes as an indicator” ofthe effect of ligands [9]. In diiron(III) complexes, anoxo bridge typically gives rise to a J value of ca. –80 to–140 cm–1 (*p–2J S17S2), while a hydroxo bridge af-fords a J value of –8 to –30 cmP1. The oxo bridge alsocan be expected to mediate electron exchange couplingin mixed-valent complexes to a greater extent that cana hydroxo bridge. The weaker coupling afforded by thehydroxo bridge facilitates electronic spin relaxation atlow temperature in the mixed-valent species throughthe Orbach-Aminov process. The spin states are morestrongly mixed when J and the Zeeman interaction aremore comparable and the gap between the ground andfirst excited spin state is small [9, 35] and more readilyexcited by the thermal spectrum of lattice vibrations orphonons. The weaker coupling also allows the zero-field splitting of the iron(II) center to produce largeranisotropies in the effective g-factor [49]. In the strongcoupling limit (hJ/Dhp1, where D is the zero-fieldsplitting of the ferrous ion), the effective g-tensor for anantiferromagnetically coupled ferrous (Sp2) and ferric(Sp5/2) ion is given by geff p7/3 gFe(III)–4/3 gFe(II) withan average effective g-value nearly 2 [49, 50, 51]. How-ever, when hJ/Dh approaches unity, the spin eigenstatesof the mixed-valent complex are mixed to very differ-ent degrees at different orientations, producing a largeanisotropy. In addition, gav decreases because the mix-ing diminishes the effect of a magnetic field relative tothat on a pure state.

Two observations further support this model. (1)The intermediate EPR properties of mixed-valent 2c,which has both an oxo and a hydroxo bridge and anintermediate J value of –57 cm–1 in the diferric state.(2) The EPR properties of phenoxo-bridged mixed-val-ent species which exhibit even larger g-anisotropiesthan the hydroxo-bridged complexes and have J valuesin the range of –5 cm–1. We note that similar differ-

ences are found in the EPR properties of the heterobi-metallic complexes [(Me3TACN)Ru(III)(m-O/OH)(m-O2CMe)2Co(III)(Me3TACN)]. The oxo-bridged spe-cies exhibits rhombic signals at gp2.41, 2.18, and 1.9,while the hydroxo-bridged derivative has much largerg-anisotropy (gp2.95, 2.3, and 1.39) [52]. These resultswere quantitatively explained by the different ligandfields produced by the m-OH and m-O ligands withhJh;100 cm–1pD. In the present case, however, thedifferences in ligand field are of minor importancewhere hJh~60 cm–1 and the differences in hJh have adominant effect.

Effect of annealing

At temperatures above 77 K, the constrained non-equilibrium configuration of the primary mixed-valentspecies relaxes toward equilibrium. The temperature atwhich relaxation occurs depends on the chemical struc-ture of the complex and on the solvent viscosity. In sol-id acetone/acetonitrile glasses, relaxation occurs, as arule, at the glass-ice transition of 112–116 K. Progres-sive annealing at 114 K of 1b, 2b, and 2d in acetone/acetonitrile glass results in small changes in line shape.In particular, annealing at 114 K for 50 s shifts the gII

features of 1b and 2d by 0.003–0.005 toward high field(Table 2). This shift can be caused by slight changes inligation geometry. In some cases, the signals decreaseby two- to threefold upon progressive annealing be-cause of dissociation into mononuclear iron complexes,as well as reaction with O2 or radical species.

Annealing 2c at 114 K for 5 min causes a gradualdisappearance of the initial EPR signal of Fig. 1c andsimultaneous growth of a new gav~2 signal with lowerg-anisotropy (Fig. 4a), resembling those for the mono-bridged oxo complexes, 1a and 1b. This new, mixed-valent species is characterized by slower spin relaxationand can be observed without visible broadening up to100 K, consistent with hydroxo-bridge cleavage to forma mono-bridged oxo complex.

More complex changes are observed in 3a, whereannealing at 115 K for 15–20 s causes the initial rhom-bic signal (Fig. 2a) to be replaced by a new axial signalobservable at 77 K with effective gkp1.93 and gIIp1.87(Fig. 4b and Table 2). This signal is similar to that re-ported for 3a generated by chemical reduction [33].However, this mixed-valent intermediate proved to beunstable in our solvent. After annealing at 115 K for3–5 min it converts into a new species with an axialEPR signal (Fig. 5a), characterized by large g-anisotro-py (gII;2.0, gkp1.57) and observable only at tempera-tures below 20 K, indicative of protonation of the m-oxo bridge. The signal resembles one reported for thestable, mixed-valent analog 4a [29]. These spectralchanges are proposed to be diagnostic of the protona-tion of a m-oxo bridge to produce a m-hydroxo-bridgedspecies [37]. This proposal is borne out in the muchwider range of dinuclear iron complexes studied here.

299

Fig. 4 EPR spectra of mixed-valent forms of 2c (a) and 3a (b) inacetone/acetonitrile glass reduced radiolytically at 77 K after an-nealing for 3 min and for 20 s, respectively, at 115 K, 100 kHzfield modulation amplitude 0.5 mT, gain 5000, 10.6 mW micro-wave power at 9.46 GHz, temperature 20 K

Fig. 5 EPR spectra of mixed-valent forms of 3a (a), 3b (b), and3c (c) in acetone/acetonitrile glass reduced radiolytically at 77 Kafter annealing for 2 min (a, c) and 1 min (b) at 115 K, 100 kHzfield modulation amplitude 1.0 mT, gain 5000, 10.6 mW micro-wave power at 9.46 GHz, temperature 7 K

New anisotropic signals (Fig. 5b, c), with a largespread in g-values, appeared after annealing the m-oxo-bridged 3b and 3c at 115 K for 2–5 min. These spectraare nearly identical to those in Fig. 3b, d for the hy-droxo-bridged analogs 4b and 4c, indicating that theoxo bridges of 3b and 3c become protonated upon an-nealing. Figure 3 shows the mixed-valent signals of 4band 4c before and after annealing at 115 K for 50–60 s.The slight increase in g-anisotropy shows that themixed-valent derivatives arising from 4b and 4c upon

cryogenic reduction have constrained non-equilibriumgeometries. Similar transitions were reported in themixed-valent form of Hr generated at 77 K [35]. In thecase of the hydroxylase component of MMO, whichcontains a m-hydroxo bridge in its native diferric form,reduction at 77 K gives rise to a fast-relaxing, mixed-valent species displaying a rhombic EPR signal withgp1.94, 1.86, and 1.79, similar to that of the mixed-val-ent state produced chemically [9, 38]. Thus, the behav-ior of radiolytically reduced iron-oxo sites in proteinscan be reproduced with model complexes.

Relevance to proteins

The results presented above show that cryogenic reduc-tion of oxo-/hydroxo-bridged dinuclear iron(III) com-plexes produces kinetically stabilized, mixed-valentspecies. The high yields of mixed-valent species allowthe application of various spectroscopic techniques,e.g., EPR, ENDOR, ESEEM, Mössbauer, MCD, etc.,for further studies. These states generally are unstableand have not been generated in useful amounts bystandard chemical reduction. The mixed-valent com-plexes formed at 77 K retain a ligation geometry closeto that of the initial diferric cluster and thus are goodstructural models of the diferric form. Most important-ly, proton transfer does not occur and bridging groupsappear to remain intact, allowing characterization ofthe diferric structure by EPR spectroscopy of themixed-valent state.

The shape of the EPR signals from the mixed-valentspecies depends critically on the m-oxo/m-hydroxobridging ligand. The low g-anisotropy is a unique signa-ture of oxo-bridged, mixed-valent species. All the pri-mary mixed-valent species originating from m-oxo-bridged diferric complexes have EPR signals with smallg-anisotropy and similar gav (Tables 1 and 2). Theirspin relaxation is sufficiently slow to permit EPR obser-vation up to 100–110 K. Similar signals are exhibited bymixed-valent species from E. coli R2 and metHr re-duced at low temperature [35, 36] and some oxo-bridged diferric complexes (1a [31], 2a [32], 3a [33])electrochemically reduced in aprotic solvents. In con-trast, hydroxo-bridged precursors give rise to mixed-valent species with larger g-anisotropy and faster relax-ation behavior, as observed for MMO, PAP, andmetHr reduced chemically at room temperature. Ac-cording to the above observations then, the significant-ly larger g-anisotropy observed for chemically reducedmetHr relative to radiolytically reduced metHr indi-cates that a proton must be taken up by the (m-oxo)dii-ron site upon chemical reduction, in agreement withother data [1, 2, 3].

The compounds studied here can be characterizedby their gavp(gxcgycgz)/3 and the uniaxiality parame-ter defined as hgz–(gxcgy)/2h/3, where gz is the compo-nent with the largest deviation from gav. When the pa-rameters from the primary species of the compounds

300

Fig. 6 Plot of gav and the uniaxiality parameter, (gz–(gxcgy)/2)/3,for mixed-valent species in Tables 1 and 2. Primary, mixed-valentm-oxo species, }, after annealing, [; primary, mixed-valent m-hy-droxo species, N, after annealing, n; primary, mixed-valent m-oxo, m-carboxylato species, L, after annealing, l; mixed-valentm-oxo, m-hydroxo species, X; primary, mixed-valent R2 andmetHr, R; mixed-valent MMO, M; and other mixed-valent dinu-clear iron proteins, c

measured here or listed in Table 1 are plotted (Fig. 6,filled symbols) they fall into two distinct regions, sepa-rated by the dotted line, depending on whether theycontain a m-oxo or a m-hydroxo bridge. After anneal-ing, most of the compounds (Fig. 6, open symbols)move to the region with smaller gav and larger uniaxial-ity parameter, indicative of protonation to form a m-hydroxo bridge. Compound 2c with both a m-oxo and am-hydroxo bridge lies near the boundary between thetwo regions. The dotted line between these two regionscorrectly separates the R2 subunit of ribonucleotide re-ductase which contains a m-oxo bridge and the m-oxomodel compounds from MMO and the other m-hy-droxo species. Although their points are adjacent toeach other, R2 and MMO are readily assignable to thetwo regions based on additional information that is dif-ficult to incorporate in this type of plot. Specifically, theprimary, mixed-valent R2 or metHr species had EPRsignals observable to about 100 K and annealing re-sulted either in loss of the mixed-valent species or inunambiguous formation of a m-hydroxo species. Themixed-valent MMO species reported are not observa-ble by EPR above about 30 K and MMO reduced ra-diolytically does not change appreciably upon anneal-ing, presumably because it already contains a m-hy-droxo bridge. It seems that either annealing or the re-duction of proteins at room temperature allows sub-stantial modification of the dinuclear iron center thatcan be characterized by careful studies of the radiolyti-cally reduced species.

Whether produced chemically or by cryogenic radio-lysis, mixed-valent sites in most proteins and modelcomplexes display EPR signals with g-values ~2 [1, 4],characteristic of the effective Sp1/2 state of an antifer-romagnetically coupled, high-spin Fe(III) (Sp5/2)high-spin Fe(II) (Sp2) pair [53, 54, 55]. However,mixed-valent sites in R2 [35, 36, 56] and 4d [30] have

Sp9/2 ground states derived from the interaction of aniron(II) and an iron(III) center by double exchange.For instance, the principal g-values of the mixed-valentcenter in E. coli R2 are 13.9, 6.7, and 5.4 [35, 36, 56].Such high-spin species have EPR spectral propertiesthat lie well outside the range exhibited by the Sp1/2species plotted in Fig. 6.

Conclusion

The radiolytic reduction of dinuclear iron(III) com-plexes in solid solution at 77 K opens up possibilitiesfor investigation of mixed-valent states. The low-tem-perature reduction of EPR-silent diferric complexesproduces paramagnetic species by introducing a singleelectron without significantly altering the coordinationenvironment of the iron atom, thus allowing generationof simple models for the mixed-valent state of proteins.In addition, EPR spectroscopy provides an efficientand simple means for distinguishing oxo from hydroxobridges based on readily observable EPR characteris-tics: the small g-anisotropy and the observation of EPRspectra up to 100 K for the m-oxo-bridged species ver-sus the large g-anisotropy and spectra observed onlybelow F25 K for the m-hydroxo-bridged species.

Acknowledgements This work was sponsored by the U.S. De-partment of Energy under contract DE-AC06-76RLO 1830, byAssociated Western Universities, Inc. Northwest Division (AWUNW) under Grant DE-FG06-89ER-75522 or DE-FG06-92RL-12451 with the U.S. Department of Energy, and by the NIHthrough Grant GM-38767. R.M.D. is grateful for participation inthe SABIT program. J.A.S. and S.A.D. acknowledge the receiptof Faculty Fellowships from the AWU NW.

References

1. Que L Jr, True AE (1990) Prog Inorg Chem 38 :98–2002. Sanders-Loehr J (1989) In: Loehr TM (ed) Iron carriers and

iron proteins. VCH, New York, pp 373–4663. Wilkins PC, Wilkins RG (1987) Coord Chem Rev

79 :195–2144. Vincent JB, Olivier-Lilley GL, Averill BA (1990) Chem Rev

90 :1447–14675. Feig AL, Lippard SJ (1994) Chem Rev 94 :759–8056. Reichard P, Ehrenberg A (1983) Science 221 :514–5197. Nordlund P, Eklund H (1993) J Mol Biol 232 :123–1648. Reichard P (1993) Science 260 :1773–17779. DeWitt JJ, Bentsen JG, Rosenzweig AC, Hedman B, Green

J, Pilkington S, Papaefthymiou GC, Dalton H, Hodgson KO,Lippard SJ (1991) J Am Chem Soc 113 :9219–9235

10. Fox BG, Froland WA, Dege JE, Lipscomb JD (1989) J BiolChem 264 :10023–10033

11. Fox BG, Liu Y, Dege JE, Lipscomb JD (1991) J Biol Chem266 :540–550

12. Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P(1993) Nature 366 :537–543

13. Sträter N, Klabunde T, Tucker P, Witzel H, Krebs B (1995)Science 268 :1489–1492

14. Fox BG, Shanklin J, Ai J, Loehr TM, Sanders-Loehr J (1994)Biochemistry 33 :12776–12786

15. Dave BC, Czernuszewicz RS, Prickril BC, Kurtz DM Jr(1994) Biochemistry 33 :3572–3576

301

16. Hanna PM, Chen Y, Chasteen ND (1991) J Biol Chem266 :886–893

17. Sun S, Chasteen ND (1994) Biochemistry 33 :15095–1510218. Kurtz DM Jr (1990) Chem Rev 90 :585–60619. Lippard SJ (1988) Angew Chem Int Ed Engl 27 :344–36120. Mashuta MS, Webb RJ, McCusker JK, Schmitt EA, Ober-

hausen KJ, Richardson JF, Buchanan RM, Hendrickson DN(1992) J Am Chem Soc 114 :3815–3827

21. Borovik AS, Papaefthymiou V, Taylor LF, Anderson OP,Que L Jr (1989) J Am Chem Soc 111 :6183–6195

22. Menage S, Que L Jr (1990) Inorg Chem 29 :4293–429723. Borovik AS, Murch BP, Que L Jr, Papaefthymiou V, Münck

E (1987) J Am Chem Soc 109 :7190–719124. Suzuki M, Oshio H, Uehara A, Endo K, Yanaga M, Kida S,

Saito K (1988) Bull Chem Soc Jpn 61 :3907–391325. Suzuki M, Uehara A, Oshio H, Endo K, Yanaga M, Kida S,

Saito K (1987) Bull Chem Soc Jpn 60 :3547–355526. Schepers K, Bremer B, Krebs B, Henkel G, Althaus E, Mosel

B, Müller-Warmuth W (1990) Angew Chem Int Ed Engl29 :531–533

27. Bernard E, Moneta W, Laugier J, Chardou-Noblat S, Deron-zier A, Tuchagues J-P, Latour J-M (1994) Angew Chem IntEd Engl 33 :887–889

28. Arena F, Floriani C, Chiesi-Villa A, Guastini C (1986) JChem Soc Chem Commun 1369–1371

29. Bossek U, Hummel H, Weyermüller T, Bill E, Wieghardt K(1995) Angew Chem Int Ed Engl 34 :2642–2645

30. Ding X-Q, Bominaar EL, Bill E, Winkler H, Trautwein AX,Drüeke S, Chaudhuri P, Wieghardt K (1990) J Chem Phys92 :178–186

31. Kadish KM, Larson G, Lexa D, Momenteau M (1975) J AmChem Soc 97 :282–288

32. Holz RC, Elgren TE, Pearce LL, Zhang JH, O’Connor CJ,Que L Jr (1993) Inorg Chem 32 :5843–5850

33. Hartman J-AR, Rardin RL, Chaudhur P, Pohl K, WieghardtK, Nuber B, Weiss J, Papaefthymiou GC, Frankel RB, Lip-pard SJ (1987) J Am Chem Soc 109 :7387–7396

34. Nivorzhkin AL, Anxolabéhère-Mallart E, Mialane P, Davy-dov R, Guilhem J, Cesario M, Audière J-P, Girerd J-J, Styr-ing S, Schussler L, Seris J-L (1997) Inorg Chem 36 :846–853

35. Davydov R, Kuprin S, Gräslund A, Ehrenberg A (1994) JAm Chem Soc 116 :11120–11128

36. Davydov RM, Davydov A, Ingemarson R, Thelander L, Eh-renberg A, Gräslund A (1997) Biochemistry 36 :9093–9100

37. Davydov RM, Menage S, Fontecave M, Gräslund A, Ehren-berg A (1997) JBIC 2 :242–255

38. Davydov A, Davydov R, Gräslund A, Lipscomb J, AnderssonKK (1997) J Biol Chem 272 :7022–7026

39. Blumenfeld LA, Burbaev DS, Davydov RM (1986) In: WelchGR (ed) The fluctuating enzyme. Wiley, New York, pp369–401

40. Ichikawa T, Kevan L, Narayana PA (1979) J Phys Chem83:3378–3381

41. Fleischer EB, Srivastava TS (1969) J Am Chem Soc91 :2403–2405

42. Gomez-Romero P, Witten EH, Reiff WM, Backes G, San-ders-Loehr J, Jameson GB (1989) J Am Chem Soc111 :9039–9047

43. Norman RE, Holz RC, Zhang JH, O’Connor CJ, Menage S,Que L Jr (1990) Inorg Chem 29 :4629–4637

44. Zang Y, Pan G, Que L Jr, Fox BG, Münck E (1994) J AmChem Soc 116 :3653–3654

45. Dong Y, Fujii H, Hendrich MP, Leising RA, Pan G, RandallCR, Wilkinson EC, Zang Y, Que L Jr, Fox BG, KauffmannK, Münck E (1995) J Am Chem Soc 117 :2778–2792

46. Armstrong WH, Spool A, Papaefthymiou GC, Frankel RB,Lippard SJ (1984) J Am Chem Soc 106 :3653–3667

47. Turowski PN, Armstrong WH, Roth ME, Lippard SJ (1990) JAm Chem Soc 112 :681–690

48. Turowski PN, Armstrong WH, Liu S, Brown SN, Lippard SJ(1994) Inorg Chem 33 :636–645

49. McCormick JM, Reem RC, Solomon EI (1991) J Am ChemSoc 113 :9066–9079

50. Rodriguez JH, Ok HN, Xia Y-M, Debrunner, PG, HinrichsBE, Meyer T, Packard NH (1996) J Phys Chem100 :6849–6862

51. Sands RH, Dunham WR (1975) Q Rev Biophys 7 :443–50452. Hotzelmann R, Wieghardt K, Ensling J, Romstedt H, Gutlich

P, Bill E, Flörke U, Haupt H-J (1992) J Am Chem Soc114 :9470–9483

53. Hagen WR (1992) Adv Inorg Chem 38 :165–22254. Bertrand P, Guigliarelli B, More C (1991) New J Chem

15:445–45455. Blumberg WE, Peisach J (1974) Arch Biochem Biophys

162 :502–51256. Hendrich MP, Elgren TE, Que L Jr (1991) Biochem Biophys

Res Commun 176 :705–71057. Atta M, Andersson KK, Ingemarson R, Thelander L, Gräs-

lund A (1991) J Am Chem Soc 116 :6429–643058. Ehrenberg A, Davydov R, Allard P, Kuprin S (1991) J Inorg

Biochem 43 :53559. Ravi N, Prickril BC, Kurtz DM Jr, Huynh BH (1993) Bio-

chemistry 32 :8487–849160. Fox BG, Hendrich MP, Surerus KK, Andersson KK, Froland

WA, Lipscomb JD, Münck E (1993) J Am Chem Soc115 :3688–3701

61. Averill BA, Davis JC, Burman S, Zirino T, Sanders-Loehr J,Loehr TM, Sage JT, Debrunner PG (1987) J Am Chem Soc109 :3760–3767

62. Day EP, David SS, Peterson J, Dunham WR, Bonvoisin JJ,Sands RH, Que L Jr (1988) J Biol Chem 263 :15561–15567