Thermodynamic and Electron Paramagnetic Resonance ...

THEJOURNAI.

OF BIOLOGICAL CHEMISTRY Vol. 256, No. 11, Issue of June 10, pp. 5577-5582, 1981 Printed m U S . A

Thermodynamic and Electron Paramagnetic Resonance Characterization of Flavin in Succinate Dehydrogenase* (Received for publication, October 4, 1980)

Tomoko Ohnishi, TsooE. King$, JohnC. Salernog, Haywood Blum, JohnR. Bowyer, and Takamitsu Maida From the Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and 12222 the $Department of Chemistry, State University of New York at Albany, New York

In all the succinate dehydrogenase preparations we have so Thermodynamicparametersofsuccinatedehydrofar reported, Center S-2 is EPR-detectable and its spinrelaxgenase flavin were determined potentiometrically from the analysis of free radical signallevels as a function of ation behavior seems tobe highly sensitive to the molecular the oxidation-reduction potential. Midpoint redox po- environment around the cluster (2, 5); a weak dipole-dipole tentials of consecutive l-electron transfer steps are interaction between Centers S-1 and S-2 was observed which -127and -31 mV at pH 7.0.Thiscorresponds to a was manifested as the relaxation enhancementof S-1 spins by stability constant of intermediate stability, 2.5 X lo-’, S-2 or as either splitting or broadening of low temperature n EPR spectra of fully reduced enzymes. The spatial relationwhich suggests flavin itself may be a converter from = 2 to n = 1 electron transfersteps. ship between Center s-1and s - 2 in different succinate dehyThe pK values of the free radical (FlH- c* F1:) and drogenasepreparationshas beendiscussedin detail ina the fully reduced form (FlH2 c* F1H-) were estimated preceding paper (5). as 8.0 k 0.2 and 7.7 & 0.2, respectively. Succinate deIn this paper, we report thermodynamic parameters and hydrogenaseflavosemiquinone elicits an EPR spectrum at g = 2.00 with a peak to peak width of 1.2 mT EPR characteristics of the succinate dehydrogenase flavin even in the protonated form, suggesting the delocali- and discuss a possible mechanism for flavin to convert the n = 2 electron transfer step to that of n = 1 in a mitochondrial zation of the unpaired electrondensity. primary dehydrogenase. A relatively short CenterS-1 to flavin A close proximity of succinate dehydrogenase flavin and iron-sulfurcluster S-1 was demonstrated based on distance is impliedby the spin-spin interaction we report, of cluster S-1 and S-2 in the the enhancement offlavin spin relaxationby CenterS- supporting our earlier assignment flavo iron-sulfur subunit. 1. EXPERIMENTALPROCEDURES

Potentiometric titration of the flavin-free radical of succinate deIt is generally accepted that the succinate dehydrogenase hydrogenase was conducted anaerobically as described by Dutton (8). molecule consists of two subunits: oneflavo iron-sulfursubunit The following redox-mediating dyes weremed selectively to minimize with M, of approximately 70,000 and one iron-sulfur subunit interference from the dye g = 2.00 signals; 1-4-naphthoquinone, of 27,000. The flavo iron-sulfur subunit contains 4 Fe, 4 S,’ duroquinone, indigo disulfonate, indigo tetrasulfonate, 2-OH-1,4and one covalently bound FAD; the iron-sulfur subunit con- naphthoquinone, phenosafranine, and safranine T; each at a final tains 4 Fe and 4 S (1-4).We have proposed the presence of concentration of 50 PM. These dyes undergo redox changes in close to n = 2 processes and variations of the dye concentration from 10 to two binuclear iron-sulfur clusters (CenterS-1 and S-2) in the 500 p~ did not affect the size of the measured g = 2.00 signal. In flavo iron-sulfur subunit and one tetranuclear cluster (Centeraddition, dye signals can be distinguished from that of the flavin free S-3) in the iron-sulfur subunit based on the EPR and therradical associated with succinate dehydrogenase based on their slower modynamic characteristics of these centers and on the corre-spin relaxation rates similar to flavodoxin g = 2.00 signals as will be lationbetween enzymic activitiestowardvarious artificial described later. Spin quantitationof the free radical signals at titration electron acceptors and functional redox components which peaks were conducted by double integration of the g = 2.005 signal, were studied with various types of soluble succinate dehydro- using the flavodoxin free radical from Peptostreptococcus elsdenni as a standard (9). EPRsamples were rapidly frozen in a 1:5 mixture of genase preparations, such as reconstitutively active BS-SDH,2 methylcyclohexane and isopentane at 81 K. The sample temperature reconstitutively inactive B-SDH, and AA-SDH(2, 5, 6). was controlled by a JEOL liquid nitrogen flow system andthe temperature was monitored by a thermocouple(Cromel-gold at 0.07% * This work wassupported by National Institutes of Health Grants iron). Flavin concentration of the succinate dehydrogenase preparaGM12202, GM16767, GM25052, and HL12576, and by National Sci- tion was determined fluorometrically according to the method of ence Foundation, Grant PCM-78-16779. The costs of publication of Wilson and King (10).Protein concentration was determined as this article were defrayed in part by the payment of page charges. described previously (2). Redox titration data were simulated using This article must therefore be hereby marked “advertisement” in the University of Pennsylvania Medical School DEC-system 10 Computer Facility. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 Present address, Department of Biology, Rensselaer Polytechnic RESULTS Institute, Troy, NY 12181. ’ Preliminary results of this study were presented at the Interna- Fig. 1 presents a potentiometric titration of the g = 2.005 tional Symposium on Frontiers of Bioenergetics (7). signal of flavin free radical in the soluble succinate dehydroThe abbreviations used are: BS-SDH,butanol-solubilized enzyme 7.0. Peak to peak ampliwith succinatepreincubation; B-SDH, butanol-solubilized without genase preparation (BS-SDH) at pH succinate; AA-SDH, alkaline-solubilized from acetone powder of mi- tude of the free radicalsignal was plotted as a function of Eh (redox potential referred to the potential of a standard hydrotochondria or submitochondrial particles; EPR, electron paramagnetic resonance; UQ, ubiquinone. gen electrode) of the enzyme solution. The bell-shaped titra-

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EPR Studies of Succinate Dehydrogenase Flavin

5578

free radical and the fully reduced form of the flavin were estimated. pK values of interest are pKs (F1H. t* Fl T) and ~ K (FlHZ R c* F1H-). As seen in Fig. 2, the ~ K value R can be obtained from the pH dependence of the midpoint potential (E,) for the overall2-electron transfer, namelyfrom the oxidized to the fully reduced form of the flavin. These E , values are obtainedempirically from the E* value of the peak position of the titration curvesa t different pH. The linein the R The straight lines figure is a theoretical curve with ~ K 7.7. with a slope of -60 mV and -30 mV/pH, respectively, which fit the curve in the lower and higher pH ranges, intersect at the ~ K HThe . pK.9 does not affect the pH dependence of E, - 200 -l o o 0 1 0 0 values. Fig. 3 gives the maximum per cent free radical concenEh (mv) tration of the potentiometric titrations as a function of pH. FIG. 1. Potentiometric titration of the flavin free radical (g Below pH 7 the maximum free radical concentration remains = 2.005) signal in succinate dehydrogenase. The enzyme (68.7 7 leads toincreasing PM flavin concentration) was titrated anaerobically in 50 mM mor- unchanged andraising the pH above pH Fig. in3. The line pholinopropanesulfonic acid buffer (pH 7.0) in the presence of the maximum free radical concentration as seen following redox mediators (each at 50 p ~ ) Duroquinone, : 2-OH-1,4- drawn in thefigure is a theoretical curve witha pKs value of naphthoquinone, 1,4-naphthoquinone, indigodisulfonate, indigotetra- 7.95 and PKRof 7.7. The computer-analyzed pH dependencies sulfonate, phenosafranine, and safranine T. EPR conditions: micro- of the midpoint potentials of the first and second 1-electron wave power, 1 milliwatt;modulationamplitude, 6.3 G; microwave transfer steps, namely Eml and Em2,are presented in Fig. 4. frequency, 9.1 GHz; time constant, 0.25 s; sample temperature, 173 K. -, experimental; - - -, theoretical curve. The maximum spin con- Below pH 7 the difference between Emland E,,, Le. -100 mV, in thehigherpHrangethe difference is centration of flavin free radical was obtained from the spin quantita- isconstantand tion of g = 2.00 signalas described under “Experimental Procedures.”

4t

5

0

6

7

8

9

pH pH

FIG. 2. Determination of ~ K from R the pH dependence of E,,, values. E , values were obtained directly from the redox potentials giving titration peaks.

FIG. 3. pH dependence of the maximum per cent free radical concentration in potentiometric titrations. Spin concentration of g = 2.00 signal and the total flavin concentration were determined as described under “Experimental Procedures” and in Ref. experimental conditions are the same as in Fig. 1.

10.

Other

tion curve gives maximum signal intensity at an Eh of-79 100, mV at pH7.0 which corresponds to theE, value of the overall Em 2-electron transfer reaction (F1 t* FlH2). We designate midpointpotentials of the fist and second electrontransfer E m1 Em2 processes as EmIand Em2, respectively (F1 c* FlH./Fl; c* FlH,/FlH-).Spinquantitation of thefreeradical signal showed that 7.3% of the total flavin (measured fluorometrically as described in Ref. 8) is found maximally in the intermediate redox state in the titration at pH 7.0. At the E,, giving rise to the peak of the titration curve, the concentrations of flavin in the oxidized and fully reduced states are equal; thus, ( K ) is readily calculated as the free radical formation constant 2.5 X The E,, and E,, valuesare calculated fromthese two parameters, namely K and E,, based on thefollowing two equations: (i)E,, - Em2 = 60 log K and (ii)E,,,] + Em2 = 2 E,,,. I The Emland Em2values at pH 7.0 obtained are -127 mV and \ -31 mV,respectively. The titration gives an experimental 7 8 9 curve (solid line) which fits relatively well to a theoretical PH curve (broken line) except that the experimental points at FIG. 4. pH dependence of E,,,Iand E,,,z, midpoint potentials both ends of the titration are morebroadly distributed. of two 1-electrontransfer steps of flavin in succinate dehydroIn order toanalyze the protonation state of reduced forms genase. Individual E,, and Ems at different pH were calculated from of flavin in the physiological pH range, the pKvalues of the the data presented in Figs. 2 and 3.

5579


gradually diminished; the free radical state is more stabilized. Fitting the theoretical curves gave optimal valuesof ~ K and K pKs as 7.7 f 0.2 and 8.0 f 0.2, respectively. Thus, at pH7.0, about 90% of the free radicalis expected to be in the neutral (protonated) form (FlH.),while at pH9.0 about 90% is in the anionic (deprotonated) form (Fl:). Fig. 5 presents the flavinfree radical in succinate dehydrogenase (BS-SDH) at pH 6.5 , , and 9.0 which were obtained by poising the enzyme at redox 6 7 8 9 potentials of -16 mV and -150 mV, respectively. The radical pH gives a g 2.00 signal with a linewidth of 1.15 mT at both pH FIG. 6. pH dependence of g = 2.00 signal size in succinate 6.5 and 9.0 where the free radicals are mostly in the protonated reduced succinate dehydrogenase.Theoretical curves for case A and deprotonated form,respectively. Both spectra are typical (- - -) and case B (-. -) of Scheme I. of flavins with wings both on the lower and higher field side A. SDH with Center S-3 (11, 12). Previously, Beinert et al. (13) reported a high level of the free radical state (about 80% at pH 7.8) in reconstitutively inactive succinate dehydrogenase preparations upon reduction with succinate. When reconstitutively active BS-SDH s-lox s - 3 0 ~ at different pH valueswe obtained was reduced with succinate much lower maximum free radical concentrations, namely, about 10% below pH 7 and 20% even at pH 9 (Fig. 6). In order to understand the widely different amounts obtained of the flavin free radicals in the succinate-reduced dehydrogenase 6. SDH withoutCenter S-3 preparations, we analyzed possible redox states of succinate dehydrogenasecomponentswhenthedehydrogenase was s - 1 ~FI. poised with a succinate and fumaratecouple which delivers 2 electrons a t a time. The high potentialiron-sulfur protein type s-lOx F' F\, S-lox FIH, iron-sulfur cluster, Center S-3 becomes extremely labile toward oxidantsin the soluble state andis EPR-detectable only (FI. = FIH. + FIT) SCHEME I. Possible redox state of succinate dehydrogenase A pH 6.5 components poised with succinate and fumarate couple (n = 2) electron donor. A and B correspond to reconstitutively active respectively. and inactive succinate dehydrogenase(SDH), "

&/I

"

I

in the reconstitutively activeform (6, 14, 15). Scheme 1 gives possible redox states of succinate dehydrogenase components upon 2-electron reduction intwo different succinate dehydrogenase systems: System A retains active Center S-3; System B contains onlyinactive Center S-3. Intermolecular redox equilibration is assumed to be much slower than the intramolecular equilibration. In case A, two different redox states (S-1 reduced, S-3 oxidized, flavin free radical) and (S-1 oxidized, S-3 reduced, flavin free radical) are the states which give rise to free radical signals among a total of six different redox states. In case B, (S-1 reduced and flavin free radical) is the only state which gives rise to the free radical signal I. 15 mT among threedifferent redox states obtainable. The maximum H free radical concentration a t different pH values can be calculated for these two cases using E,,,, and Em2values of the flavin as reported above andE,,, values of 0 mV and 60 mV for Center S-1 and S-3, respectively. This is plotted in Fig. 6 with a dashed line for case B and dotted-dashed line for case A. High free radical concentrations reported for the reconstitutively inactive succinate dehydrogenase preparations(13) fits very well with case B. Data on BS-SDH fit ratherwell to the calculated curve in the pH range below 8, but gives slightly higher radical concentrations above pH 8 (Fig. 6). This is explicable from the partial inactivationof Center S-3 even in the reconstitutively active BS-SDH.It is worth pointing out FIG. 5. EPR spectra of BS-SDH at pH 6.5 and 9.0. The enzymes that if we titrate Center S-1 and flavin in succinate dehydrowere potentiometricallypoised at -16 mV and -150 mV, respectively, genase preparationswherenoCenter S-3 is reactive,the for pH 6.5 and 9.0 systems. The former was dissolved in 7 mM 4-(2- titration with a succinate/fumarate couple gives the same hydroxyethy1)-1-piperazineethanesulfonicacid buffer and 43 mM 2- redox titration curve for these two components, an interme(N-morpho1ino)ethanesulfonicacid buffer (pH 6.5) at the final flavin concentration of 63.9 PM and the latter was dissolved in 50 mM N - diate n value between 1 and 2 below the midpoint potential, and approximately n = 1 curve in the higher potential range tris(hydroxymethy1)methylethanesulfonicacid bufferat 3 0 . 9 ~EPR ~. sample temperature, 213 K; input microwave power, 100 microwatts, of the midpoint. modulation amplitude, 6.3 X tesla. In order to study spatial relationships between flavin and


5580

Center S-1 in succinate dehydrogenase, we comparedthe power saturation behavior of the g = 2.00 signals of the flavin free radicalof succinate dehydrogenasewith that of flavodoxin a t twodifferent temperatures. In flavodoxin, no transition metal ion is present in the vicinity of the flavin free radical (7). A saturationparameter,wasdetermined according to the quantitative procedure reported by Blum and Ohnishi (16). P 1 / 2 is the input microwave power level at which the saturation condition is satisfied as discussed in Ref. 16. The Pllnvalue of succinate dehydrogenase flavin is significantly higher than thatof flavodoxin both at 193 and at 233 K. It is interesting to see that the spin relaxation of the flavodoxin free radical is insensitive to the temperature difference of 40 K in the above temperature range, whereas the PI/*value of the succinate dehydrogenaseflavin free radical signal is doubled by raising the temperature from 193 K to 233 K. This indicates that in the succinate dehydrogenasemolecule, spin relaxation of the flavin free radical signal is enhanced by S-1 effect, flavin nearby CenterS-1 spins. In order to test this spin relaxation was examined after complete destruction of Center S-1 by lowering the pH of the enzyme to 4 and then returning to neutral pH. As seen in Table I, spin relaxationof the succinate dehydrogenase flavin approaches that of the flavodoxin free radical in the absence of S-1 spins. These results suggest a small distance between flavin and at least A (233K)

P , , p 20 llm

0.

.

0

aa

0.1

i

0

i n p n M i c r w n Powr (mW)

FIG. 7. Power saturation behaviorof the flavin free radical in succinate dehydrogenase and flavodoxin at two different temperatures.

TABLE I Saturation parameter (P1,2) ofthe flavin free radical in the presence and absence of Center S-1 spins Flavodoxin free radical was produced by illumination in the presence of EDTA as described under “Experimental Procedures.” Succinate dehydrogenase free radicals were obtained potentiometrically in the case of B-SDH, and by succinate addition for B-SDH. To destroy cluster S-1, the pH of the enzyme was lowered to pH 4.0 by the addition of sodium acetate and then brought back to neutral pH. EPR conditions are the same as in Fig. 1 except input microwave power levels. EPR sample temperature was 170 K. Samples

A. B. C. D.

Flavodoxin BS-SDH 0.78 poised mVat -78 B-SDH reduced with succinate Acid-treated BS-SDH poised at mV -70

PIp2 values milliwatts

0.20 0.80 0.23

one of the binuclear iron-sulfur clusters, namely Center S-1. Center S-2 is not paramagnetic under the experimentalconditions required for samples shown in TableI. DISCUSSION

The presentstudyshowsthatsuccinatedehydrogenase flavin has a free radical formation constantof K = 2.5 X at neutral pH which is a far more stable intermediate redox state than the typical n = 2 redox components, suchas a free ubiquinone/ubiquinol couple in a hydrophobic milieu ( K = 10”’) (17),or the NAD+/NADHcouple ( K = lo-”) (18). This indicates that succinate dehydrogenase flavin itself may function as a good converter from an n = 2 to an n = 1 electron transfer process. The flavin accepts electrons from succinate in a 2-electron step: theflavosemiquinone is stable enough to permit the reduction of iron-sulfur clustersof the dehydrogenase in sequential 1-electron steps, although the detailed mechanism of the latter steps is not yet completely known. It is therefore unnecessary to postulate simultaneous reduction of two iron-sulfur clusters by flavin as an n = 2 to n = 1 stepdown mechanism (19). Recently, Albracht proposed that succinatedehydrogenase contains only one binuclear (Center S-1)and one tetranuclear iron-sulfur cluster (CenterS-3) per molecule based mostly on the nondetectability of the Center S-2 EPRsignal in a succinate-cytochrome c reductase preparation (20) as well as in succinate dehydrogenasewhich was isolatedfrom Complex I1 and retains almostfull reconstitutive activityin a highlypure form (4,21). We have c o n f i i e d these experimental observat i o n ~Even . ~ in these two systems, spin relaxation of Center S1 is dramatically enhanced by fully reducing the enzyme with dithionite. Albracht (20) interpreted this spin relaxation enhancementas being caused by a proteinconformational change induced by the reduction of the flavin to the fully reduced state. The enhancement of the S-1spin relaxation is n = 1 redox component(CenterS-2) with causedbyan midpointpotential of approximately -400 mV insoluble succinate dehydrogenase preparations (2, 6). Analysis of the titration data presented in Fig. 1 indicates that a midpoint potential (E,) of -81 mV would be obtained for a titration monitoring the fully reduced formof flavin, and -77 mV for a titration of the oxidized form, both withn value close to 2. Neither midpoint potential nor n value for the flavin reduction support the hypothesis proposed by Albracht (20) and the enhancementof S-1 spin relaxation seems to be consistent with thecross-relaxation of S-1 via S-2 spins (2, 5, 6). As we reported previously, spin relaxation of Center S-2 greatly depends on the microenvironment of the active center. We interpret the nondetectability of Center S-2 in these intact systems as being due to either (i)extremely an short relaxation time for S-2 spins or (ii) the spin coupling between S-1and S2 is such thatwe do not seesignals formore than one spin per molecule in the fully reduced enzyme of these intact preparations. The presence of two binuclear and one tetranuclear ironsulfur clusters in the succinate dehydrogenase molecule has also been demonstratedby an independentprocedure, namely, iron-sulfur core extrusion and core displacement as reported by Coles et al. (4). Ackrell et al. (22) conducted a potentiometric titration of the activationprocess of the enzyme in the presence of excess oxalacetate; oxalacetate binds tightly to succinate dehydrogenase in the oxidized state, inactivating the dehydrogenase (22). In this method an oxidation-reduction component was Em7.0value between -60 and titrated with midpoint potential -90 mV in an n = 2 process, suggesting the titration of the T. Ohnishi, H. Blum, C. A. Yu, and L. Yu, unpublished data.

EPR Studies of Succinate Dehydrogenase succinate dehydrogenase flavin. This is in good agreement with the titrationof the fully reduced form of flavin. In this paper we have demonstrated the close proximity between succinate dehydrogenaseflavin and Center s-1from the enhancementof flavin spin relaxationby S-1spins (Fig. 7 and Table I). We previously reported that the distance between the S-l and S-2 clusters is approximately 10 A (2, 5 ) ; a high potential iron-sulfur protein-type cluster (Center s-3) and a ubiquinone pair (23, 24) which is the specific electron acceptor of succinate dehydrogenase are also considered to be located within about15 A of each other (24, 25). As shown in Fig. 7 in this paper, the saturation parameter ( P I / Pof) succinate dehydrogenaseflavin is 2.0 milliwatts at 233 K, indicating that there is an iron-sulfur cluster in the nearvicinity of the flavin. This is a binuclear rather than tetranuclear cluster, since intact Center S-3 is present in BS-SDH and absent in B-SDHanddestruction of S-1 and S-2 causesthe flavin relaxation to slow markedly. The very short relaxation time (Pl12> 100 milliwatts at 233 K) observed with the g = 2.00 signal of ubisemiquinone is consistent with its location close to the tetranuclear cluster, Center S-3 (24-26). These results further strengthen our earlier topographical assignment of S-1and S-2 in flavin-iron-sulfur subunit two binuclear clusters and Center S-3 tetranuclear iron-sulfur cluster in the ironsulfur subunit. Using the previously reported lifetime broadening of the Center S-1 EPR spectrum (27), we can estimate the TI of s a t 230 K. The flavin power satuCenter S-1 as about ration curve in the presenceof reduced Center S-1 has a of about 2 milliwatts, which corresponds toa cross-relaxation time of the orderof 1 O ” j s. Since nobroadening or splittingof the iron-sulfur EPR spectrum due to theflavin semiquinone can be observed at low temperature, we can place an upper limit of t 5 G on any coupling between the two. We thus expect a flavin-iron-sulfur distance of at least 12 A. In the absenceof spin-spin interaction,a system containing two S = ?hspecies in a static magnetic field fi,, will have four states, aa, ab, pa, and pp, which correspond to the possible orientations of eachspininthemagnetic field (“up up,” “updown” etc.). The Zeeman interactions can be represented

Flavin

5581

first order splitting (of 2A for the dipolar terms) but do not mix the states. The terms in SI’SZ-and SI-&’ connect the up and pa states. These states aremixed and two new states result $2

= clap

4.1 =

+ c$a

c , p a - clap

If the coupling terms are smaller than the energy difference between ap and Pa, ( ( g l - g ) P H o ) c ~will be nearly 1, and , B‘ is using perturbation theory el B’/(gl - g z ) p H ~where the sum of the dipolar B and exchange terms. The S2 transitions thus have someSI character even with relatively weak coupling if the separation between ap and pa is smaller (gl g z ) . In the limiting case, ap and pa are degenerate and the states are completely mixed (e1 = e2 = l/d2). The fast relaxation of SItransitions must resultfrom matrix and elementsfromothersources whichcouple aa with ap with Pp. Inthe coupled system,there will be matrix elements of this type connecting aa with $2 and 4.1 with ,8/3 weaker by a factor of B ’ / ( g l - g ~ ) p H than , ) those connecting aa and pa in the uncoupled system. The transition probability depends on the squareof the matrix elementconnecting two states. Thus, we expect the predominantly SP transition to acquire a new relaxation mechanismwith acharacteristic time l/TI.%= l/Tls,-(-B’/(gl - &PHo)’. in the dipolar andexchange HamiltonThe termsin SI,Szz ians give rise to the first order splittings; clearly, these must be withinthe linewidth. The terms in S1+S2-and Sl-S2’ connect theup and pa states. This contributes to observed the splitting when the magnitudeof the off diagonal terms is not much smaller than the separationof the basis states. Since theg tensors of both theiron-sulfur cluster andflavin semiquinone are not very anisotropic andoverlap, rather small dipolar orexchange couplingswould be effective in mixing the SI’Sz- and flavin and iron-sulfur transitionsthroughthe SI-Sn’ terms. The anisotropy of the iron-sulfur center, while not large in proportion to its magnetic moment, will lead to an angular dependent & = gl - g2.At some orientations, gl = g2 and the iron-sulfur and flavin semiquinone transitions will be completely mixed. It will be difficult to saturate the “flavin” transition of molecules oriented in Ho so that gl and H,een,,,, = ( S ~ * ~ I . H+O S , . g d . H o ) g2are nearly equal. The energies of the four states are thus The majority of molecules will not have these special oriL.’L(gl+ gdPHC1, %(gl- gdPHo, -Wgl - gdPHcIand -%(gI+ gz)pHu entations. The onset of saturation will bedetermined by molecules which have (g, - g z [ nearly maximal or have the If S I has a rapid relaxation rate but S2 relaxation is slow, value of the B’ term near zerobecause of the (1-3 cos%) the aa ++ pa and ap t, pp transitions will be difficult to dipolar angular dependence; the overall saturation behavior saturate while aa t,ap and pa t,pp will be easily saturable. will be morecharacteristic of theaverageseparationand The dipolar interaction betweentwo spins S I and S I can be average B’ value. A full description would require numerical represented by the Hamiltonian integration over all orientations a t each power. This would depend on the orientation of the g tensors with respect to g&?S,. s, - glg@*(S, .F) (S,.F) H