[17] Combining Mössbauer spectroscopy with integer spin electron paramagnetic resonance

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

463

Conclusions Multifield saturation magnetization studies of metalloproteins have been rare until now. This can be expected to change. A fully automated susceptometer with the requisite sensitivity is commercially available. Software required for fitting the data is commercially available, as are inexpensive high-speed computers capable of thoroughly analyzing a given data set. The fundamental techniques for identifying and minimizing background signals have been discussed in this chapter. With these tools and procedures in place, we can expect increasing application of this new methodology. This methodology is unique in detecting all of the magnetic sites in a given sample. Proper interpretation of multifield saturation magnetization data, however, can still be tricky. Good judgment is required to deal properly with impurities. The technique does not lend itself to exploratory work on magnetically complicated systems because the methodology lacks the resolution of resonance techniques. Even so, several "discoveries" have been made using this methodology after thorough measurements with EPR and M6ssbauer spectroscopies had laid the groundwork. Saturation magnetization measurements were then able to answer a single, well-defined question that remained. Acknowledgments I thank Jim Peterson who contributedto the methodology,ThomasA. Kent who made helpful critical commentson the manuscript, and Mariana S. Sendova who drew Fig. 5. This research was supported by National Institutes of Health Grant GM32394.

[1 7] C o m b i n i n g M 6 s s b a u e r S p e c t r o s c o p y w i t h I n t e g e r S p i n Electron Paramagnetic Resonance

By

ECKARD MONCK, KRISTENE K. SURERUS,

and

MICHAEL P. HENDRICH

Introduction In 1978 we wrote an article in this series about 57Fe M6ssbauer spectroscopy of electron carrier proteins ~that focused on the relationship between M6ssbauer spectroscopy and electron paramagnetic resonance (EPR). Since then, these correlations have been frequently utilized to untangle 1 E. M0nck, this series, Vol. 54 [20].

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by Academic Press, Inc. All fights of reproduction in any form reserved.

464

PROBES OF METAL ION ENVIRONMENTS

[17]

the complex MOssbauer spectra of proteins with multiple Fe sites and have played a crucial role in the discovery of Fe3S 4 clusters. 2 Exploitation of the connection between these two techniques has also provided fundamental insights into the nature of protein active sites. Thus, MOssbauer and EPR studies lead to the conclusion that the active site of Escherichia coli sulfite reductase contains a siroheme prosthetic group covalently linked to a Fe4S 4 cluster. 3 This conclusion has subsequently been confirmed by X-ray crystallographic studies. Owing to the utility of combining MOssbauer and EPR studies, we are prompted to further develop this theme here. The previous article ~also described the benefits of studying the MOssbauer spectra of compounds with integer electronic spin (non-Kramers systems) in strong applied magnetic fields. Such studies, described for the S = 2 state of reduced cytochrome P-450, were just emerging as a powerful probe of the electronic structure of non-Kramers systems. As metals in states with integer spin were not suspected to yield EPR signals, a useful connection between EPR and MOssbauer spectroscopy was apparently nonexistent. However, more recent observations have changed this picture considerably. 4-s Thus, integer spin EPR signals have been detected and analyzed for many metalloproteins and synthetic complexes s that mimic active site structures. Not surprisingly, there are close correlations between the MOssbauer and EPR spectra of integer spin systems, just as previously shown for Kramers systems. Moreover, newly developed methods for the analysis of the integer spin EPR line shape 4-8 have made it feasible to determine spin concentrations with a precision approaching that achieved for Kramers systems. In this chapter we further describe the correlations between MOssbauer and EPR spectroscopies and indicate the potential for these methods for the quantitative analysis of non-Kramers systems. Throughout this chapter we assume that the reader is familiar with the concepts and nomenclature outlined in the previous account on MOssbauer spectroscopy. 2 M. H. Emptage, T. A. Kent, B. H. Huynh, J. Rawlings, W. H. Orme-Johnson, and E. Miinck, J. Biol. Chem. 255, 1793 (1980). J. A. Christner, E. M0nck, P. A. Janick, and L. M. Siegel, J. Biol. Chem. 256, 2098 (1981). 4 M. P. Hendrich and P. G. Debrunner, Biophys. J. 56, 489 (1989). 5 M. P. Hendrich, E. Miinck, B. G. Fox, and J. D. Lipscomb, J. Am. Chem. Soc. 112, 5861 (1990). 6 M. P. Hendrich, L. L. Pearce, L. Que, Jr., N. D. Chasteen, and E. P. Day, J. Am. Chem. Soc. 113, 3039 (1991). K. K. Surerus, M. P. Hendrich, P. D. Christie, D. Rottgardt, W. H. Orme-Johnson, and E. Miinck, J. Am. Chem. Soc. 114, 8579 (1992). s C. Juarez-Garcia, M. P. Hendrich, T. R. Holman, L. Que, and E. MOnck, J. Am. Chem. Soc. 11.3, 518 (1991).

[17]

COMBINING MOSSBAUERAND EPR SPECTROSCOPIES

465

M6ssbauer and Electron Paramagnetic Resonance Spectroscopy of Non-Kramers Systems In this section we describe the connections between M6ssbauer and EPR spectroscopy for systems with integer electronic spin. For mononuclear systems the state of most interest is the high-spin (S = 2) ferrous ion. However, the considerations developed below apply also to highspin (S = 2) Fe 4+ and to metal clusters with other spins such as observed in iron-oxo proteins (hemerythrin, ribonucleotide reductase) and nitrogenase (the P cluster in the state pox). The discussion is best framed in the spin Hamiltonian formalism, and to be as concrete as possible we discuss the specific case of an S = 2 system. The electronic part of the pertinent spin Hamiltonian is commonly written as 'g = D

Sz 2 -

S ( S + 1) + ~ (Sx 2 - Sy 2)

-b/3S.g. H

(1)

Here D and E are the axial and rhombic zero-field splitting (ZFS) parameters, respectively, and g is the electronic g tensor. For a sample with randomly oriented molecules, such as protein in frozen solution, the orientation of the electronic x, y, z coordinate system of Eq. (1) relative to a crystallographic coordinate system is usually not known. Such information can only come from spectroscopic studies of single crystals. However, one can always choose an electronic coordinate system x, y, z, such that the parameter E / D is confined to 0 -< E / D <- ~. For simplicity, we assume that the orientations of the ZFS and g tensors are the same. For highspin Fe z+ the principal values o f g are generally confined to 2.0 < gi < 2.3. Figure 1 shows, for D < 0, the zero-field energies resulting from Eq. (I) plotted against E / D . In the following we focus on the two lowest levels. Using the standard representation IS,M) = I2,M) -= [M), the zero-field eigenstates and the associated energies of these two levels can be written a s 4 [2+) = a+([+2) + I-2))/21/2 + a-10) [2-) = ( 1 + 2 ) - I-2))/2 '/2

E+ = - 2 ( 0 2 + 3E2) 1/2 E_ = - 2 D (2)

where a -+ ~ {1 --- [1 - ~(E/D)2]}1/2/21/2. The energy difference between the two levels, A = E_ - E + , can be conveniently approximated as A -~ 3 D ( E / D ) z. Now, if a is smaller than the microwave quantum (hv 0.3 cm-1 at X-band) one may observe an EPR transition between the two states. The probabilities for EPR transitions are obtained by standard procedures, namely, by computing the matrix elements of [(2+[13S • g. H~]2-) 2, where H1 is the magnetic component of the microwave field.

466

[17]

PROBES OF METAL ION ENVIRONMENTS

M=..~0

"-'-

2D

D

S=2

~ ,

0

M=+2

-2D

0

0.05

~

0.1

0.15 E/D

0.2

i

12->

i

,

I

i

0.25 H ('13

i

i

T

I

0.5

12->

0.25

0.3

FiG. 1. Energy levels of an S = 2 system for H = 0 and D < 0 according to Eq. (1). F o r D = - 1.8 cm -] and E/D = 0.23 an EPR transition between the two lowest levels (A = 0.28 cm -l) can be observed at X-band frequencies (hv ~- 0.3 cm-l). For E = 0, and with an applied field along z, the levels can be labeled with the magnetic quantum n u m b e r M. The inset s h o w s the EPR transition between the [2+>and [2-) levels.

For systems with half-integral electronic spin only the matrix elements involving Sx and Sy are nonzero, and thus the selection rules for EPR transitions are AM = ___1. For the levels considered here, on the other hand, only the matrix element involving S z is finite; (2÷rSz[2 -) = 2a ÷ because Sz([+2) - I - 2 ) ) = 2([+2) + I - 2 ) ) a n d <0[Szl2-> = 0. Consequently, only the component of 1-I1 fluctuating along z is effective. These expressions show that the EPR transition between the [2 +> and 12-) levels has AM = 0 (not AM = ---2, --+4 as frequently described in the literature).

[17]

COMBINING Mt)SSBAUER AND E P R SPECTROSCOPIES

467

However, transitions between levels of an integer spin system do not necessarily have AM = 0. For instance, if we choose values of D and E such that the two highest levels of Fig. 1 are split in energy by less than 0.3 cm -1, a AM = +-1 transition can occur. We have analyzed such a transition for an S = 3 system. 8 In the presence of a static magnetic field the 12+-) levels split quadratically as shown in the inset of Fig. 1. This nonlinear field dependence is due to a competition between the zero-field and Zeeman interactions to define the direction of the magnetic moment. In contrast, levels of halfintegral spin systems split linearly with the magnetic field (for flH ~ D) because the direction of the magnetic moment is defined by the magnetic field alone. As a result, the resonance condition for integer spin doublets with the field along the z axis is (hv) 2 = A2 + (geff]3H) 2

(3)

where geff = 4a+gz is an effective g value and where gz is the component of the g tensor of Eq. (1); for E/D < 0.15 the effective g value is near 8. The above-mentioned fundamental differences between integer and halfintegral spin systems give rise to dramatically different EPR spectra. To give the reader a flavor for the variety of integer spin EPR resonances, we have displayed in Fig. 2 traces of spectra obtained for nitrogenase, cytochrome-c oxidase, and the hydroxylase of methane monooxygenase. Two characteristic features readily distinguish the spectra of integer from half-integral spin metal species. First, as a consequence of the selection rule AM = 0, integer spin signals grow in intensity when H1 is oriented parallel to H. Second, because doublets of integer spin systems are split in zero field by A which may be near A = 0.3 cm-1, EPR resonances may be observed near H = 0. The interpretation of integer spin EPR spectra is also quite different from that of half-integral spin spectra. The assignment of a signal to a specific metal species requires consideration of all aspects of the spectrum and not just the position of the resonance alone. The nonlinear field dependence causes highly skewed line shapes. Moreover, a nonzero value of A will result in a shift of the resonance to lower field, away from geff. Thus, marking the g value of some feature of the spectrum is of marginal value, since that g value is not equal to g~tr of Eq. (3). Hendrich and Debrunner 4 have discussed the orientation dependence of the EPR resonances, and they have described procedures for fitting the line shapes of the spectra. Data obtained for a variety of metalloproteins and synthetic complexes have shown that heterogeneity in the local environment of the metal often leads to substantial broadening of the resonances. This heterogeneity should not be confused with "sample

468

[17]

PROBES OF METAL ION ENVIRONMENTS g = 15.5

I

.._._

~

A 9.3

f

a

"r

? 16.0

"¢3

I

xO.5

-2/ I

0

I

!

1 O0

C

I

I

200

300

H (mT) FIG. 2. X-band (9.1 GHz) EPR spectra of the P clusters of nitrogenase 7 (A), beef heart cytochrome-c oxidase (B), 4 and the diferrous hydroxylase of methane monooxygenase (C). 5 The spectra were recorded in parallel mode, that is, the magnetic component of the microwave field was fluctuating parallel to the static field. The spectra are plotted for equal instrumental conditions (T = 4 K) and sample concentrations (0.2 mM).

heterogeneities" that biochemists laboriously try to remove by using, for example, column techniques. The latter type of heterogeneity may arise from denatured protein or protein isozymes. The heterogeneity discussed here is an intrinsic property of the metal site; it may arise from a continuum of conformational subsites of the protein lattice or from random solvent-protein interactions. These heterogeneities affect the orbital states and, via spin-orbit coupling, cause the ZFS parameters D and E to be distributed. This, in turn, yields a distribution of A in Eq. (3). Without adequate treatment of the line shapes (by computer simulations), quantitation of the spin concentration, a major goal in biochemical EPR spectroscopy, is not possible. For the discussion of the Mrssbauer spectra we amend Eq. (1) with terms describing the hyperfine interaction of the 57Fe nucleus with the electronic environment:

~hf

=

S" A" I + I" P" I - g~8,H" I

(4)

where S . A . I describes the magnetic hyperfine interactions, and -gn~n H" I is the nuclear Zeeman term. The I. P . I term describes the interaction of the quadrupole moment, Q, of the nuclear excited state ( / = ]) with the electric field gradient tensor [principal components Vxx,

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

Vyy, Vz~ ; r~ = (Vxx usually written as ~Q =

469

Vyy)/Vz~]. In its principal axis form this term is

eQVzz 1---~[31z2 - I(I + 1) + rt(Ix2 - Iy2)]

(5)

In the absence of magnetic effects, the M6ssbauer spectrum of an iron site generally consists of a two-line pattern, a quadrupole doublet, the splitting of which is given by AEQ =

2

1 +-

(6)

As discussed previously, 1 the magnetic splitting observed in a M6ssbauer spectrum is determined by the effective magnetic field He~r = Hin t hH, where Hin t =

- (S}. A/gnfl n

(7)

is the internal magnetic field and where (S) is an appropriately taken expectation value of the electronic spin S. Because we previously ~ discussed the meaning and applications of Eq. (7) in considerable detail, we do not dwell on it here. Let us focus again on the two lowest levels of the S = 2 system and assume that we conduct M6ssbauer experiments at temperatures where the other three states are not populated. (This restriction is not essential for the following arguments.) We assume that the electronic spin relaxation rate is slow on the M6ssbauer time scale (-10 MHz); for metalloproteins this condition is usually fulfilled at T < 4.2 K. In the slow relaxation limit each spin level of Eq. (1) contributes its own M6ssbauer spectrum. Therefore, we need to compute (S) for each electronic level. Using the eigenstates given in Eq. (2) the reader may verify that (Si) = 0 for the two levels, in accord with the general result that singlet states do not have a permanent moment, - fig. (S), in the absence of an external magnetic field. An applied field mixes the two electronic states through the Zeeman term of Eq. (1). The mixing is proportional to the matrix elements of the Zeeman interaction taken between the two electronic levels, that is, proportional to (2+[S/[2-). These are the same elements which control the EPR transition probability! Because (2+1Sxl2 -) ~ (2+lsrl2 -) ~ 0 and (2+1Szl2 - } = 2a + ~ 2, mixing is most effective for those molecules of the sample that have their z axis, defined by Eq. (1), parallel to the applied field. In Fig. 3 we have plotted (solid lines) the expectation values (Sx), (Sy), and
470

PgOBES OF METAL ION ENVIRONMENTS

[17]

1.9

1.4

i

0.9

0.4

Y -0.1

,

0

,

,

,

I

0.1

J

,

,

.

I

.

.

.

.

0.2

I

0.3

.

.

.

.

I

0.4

.

.

.

.

0.5

H (T) FIG. 3. Solid curves give expectation values (Sx),
y c o m p o n e n t s result f r o m mixing with the M = ---1 levels o f the S = 2 manifold. T h e 12-) state will p r o d u c e a similar graph, e x c e p t that its (Sz) value is positive. A t l o w fields, (S z) o f Fig. 3 has the slope -8flgza+2/A. F o r small values o f A the e x p e c t a t i o n value (Sz) saturates easily, and the M O s s b a u e r s p e c t r u m exhibits its m a x i m a l H i n t = -(Sz)Az/(gnl~n) at low applied field. T h u s , if o n e o b s e r v e s the full internal field in applied fields b e l o w 0.5 T, o n e c a n infer that the electronic splitting A is such that E P R transitions are o b s e r v e d at X - b a n d ; w e a d d r e s s the limit A ~ 0 below. B e c a u s e an E P R - a c t i v e state p r o v i d e s additional s p e c t r o s c o p i c a c c e s s to the s y s t e m u n d e r study, this is i m p o r t a n t i n f o r m a t i o n for the biochemist. E v e n w i t h o u t a detailed u n d e r s t a n d i n g o f the line shape, the signal amplitude c o n v e y s i m p o r t a n t i n f o r m a t i o n in r e d o x titrations and substrate binding studies.

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

471

Figure 4 shows theoretical M6ssbauer spectra computed along the "magnetization" curve of Fig. 3 for those values of H indicated by the circles on the (Sz) curve. Note that the magnetic splitting increases rapidly with increasing applied field and that the final six-line pattern has developed essentially for H = 0.5 T. From such patterns the M6ssbauer spectroscopist can construct the (Sz) curve of Fig. 3 and also deduce that (Sx) and (Sy) are small. This knowledge, in turn, allows one to determine the ZFS parameters D and E/D and to predict whether an EPR signal can be observed at S-, X-, or Q-band. Conversely, the observation of an integer [

l

l

l

I

I

l

l

l

l

i

l

l

l

l

1.0T

0.5 T

0.2T

0.1T

0.05 T

O.OT

I

-6

l

-4

I

I

-2 0 2 VELOCITY (mm/sec)

I

I

I

I

I

4

I

I

6

FIG. 4. Theoretical M6ssbauer spectra o f the 12+) level of Fig. 1 computed along the "magnetization" curve of Fig. 3. In zero field the spectrum consists of a quadrupole doublet. The spectra originating from the 12-) level are very similar to those of the 12+) state.

472

PROBES OF METAL ION ENVIRONMENTS

[17]

spin EPR signal gives considerable insight regarding the general features of the M6ssbauer spectrum. This knowledge may allow one to correlate a M6ssbauer component with the EPR-active species for metalloproteins containing more than one metal center with integer electronic spin.

Example: Desulfovibrio gigas Ferredoxin II Ferredoxin II (Fd II) from Desulfovibrio gigas is an electron transfer protein that has been studied with a variety of spectroscopic techniques. The protein contains an Fe3S 4 cluster that can be stabilized in two oxidation states. 9 In the oxidized state, the cluster contains three high-spin ferric sites that are antiferromagnetically coupled to yield an S = ½ground state. The X-ray structure of oxidized Fd II has revealed that, in accord with the spectroscopic data, the Fe3S 4 cluster has a structure like the cubane Fe4S4 clusters except that one Fe site is unoccupied.l° The reduced Fe3S4 cluster has a ground state with electronic spin S = 2. The M6ssbauer spectra of this state have been analyzed in detail by Papaefthymiou et al.,9 and in the following we describe some of their results in light of the discussion of the preceding section. These authors have also reported an EPR spectrum of reduced Fd II; here we provide spectral simulations for these data. Figure 5 shows M6ssbauer spectra of Fd II recorded in zero field (curve C) and in applied fields of 0.25 (curve B) and 1.0 T (curve A). The zero-field spectrum consists of two quadrupole doublets, labeled I and II, with a 2:1 intensity ratio. Doublet I has AEQ = 1.47 mm/sec and an isomer shift 8 of 0.46 mm/sec. The two identical sites associated with this doublet constitute a valence delocalized Fe2÷-Fe 3÷ pair. Site II, on the other hand, has parameters typical of high-spin Fe 3+ with tetrahedral sulfur ligation (AEQ = 0.52 mm/sec and 8 = 0.32 mm/sec). Analysis9 of a large set of spectra recorded in strong applied fields yielded D = - 2.5 cm -~ and E/D = 0.23. The graph shown in Fig. 3 was constructed with these parameters. The arguments presented in the preceding section all apply to the analysis of the Fd I1 spectra, except that we have to compute one M6ssbauer spectrum for each distinct Fe site. (The spectra of the delocalized pair were found to be indistinguishable under all experimental conditions.)

9 V. Papaefthymiou, J.-J. Girerd, I. Moura, J. J. G. Moura, and E. MOnck, J. A m . Chem. Soc. 109, 4703 (1987). J0 C. R. Kissinger, E. T. Adman, L. C. Sieker, and L. H. Jensen, J. A m . Chem. Soc. 1109 8721 (1988).

[17]

C O M B I N I N MOSSBAUER G AND EPR SPECTROSCOPIES i

l

l

l

l

l

l

l

l

l

l

l

II

473

l

V f--

0.0 0.5

r" A= 1

.

0

T

~

Z 0 0.0 0 r~

< 0.5

H =0.25 T

0.0 2.0

C

4.0

I

-6

I

I

-4

I

I

I

-2 0 2 VELOCITY (mm/sec)

I

I

I

I

t

4

I

I

6

FIG. 5. Low-temperature M6ssbauer spectra of the S = 2 state of the Fe3S4 cluster of reduced D. gigas Fd II. The zero-field spectrum (4.2 K, C) consists of two doublets with 2 : 1 intensity ratio. Spectra shown in (A) and (B) were recorded in parallel applied fields at 1.5 K. The theoretical spectra displayed above the 1.0 T spectrum show the contributions of the delocalized Fe2+-Fe3÷ pair (site I) and the localized Fe 3+ (site II),

Figure 6 shows two EPR spectra of Fd II recorded at 4.2 K with a bimodal cavity. For recording trace A in Fig. 6 the cavity was tuned such that the microwave field H1 was fluctuating parallel to the static field H (parallel mode), whereas trace B was obtained in the standard transverse

474

PROBES OF METAL ION ENVIRONMENTS

[17]

g=18

I

A

S

" ~ . . . . ~.~

-1-

"10 =

I

0

I

1oo

I

I

200

I

I

300

I

400

H (mT) FIG. 6. X-band EPR spectra of the S = 2 state of the Fe3S4 cluster of reduced D. gigas Fd II recorded at 4 K in parallel (A) and perpendicular (B) mode (M. P. Hendrich, I. Moura, J. J. G. Moura, K. K. Surerus, E. M~nck, unpublished results). The dashed curves are theoretical spectra computed with the parameters quoted in the text.

mode. Note that the minima of the resonances do not occur at the same field in the two spectra, showing that one cannot obtain the effective g value by simply marking the minima of the curves. The dashed lines are the result of computer simulations using the S -- 2 Hamiltonian of Eq. (1) with D = - 2.5 c m - ~, E/D = 0.23, and g = (2.0, 2.0, 2.0). To produce the correct shapes of the spectra we have assumed that the parameter E/D has a Gaussian distribution centered around the mean E/D = 0.23 with trE/D = 0.055. The quoted parameters yield a mean A of 0.38 cm-1 and a A distribution as shown in Fig. 7. The amplitude of the theoretical curve in Fig. 6A was adjusted to fit the experimental curve. Absolute spin quantitation of the sample relative to a standard, Fe(II)-doped zinc fluorosilicate, gave a spin concentration of 6.6 mM S = 2 spin, which compares well with the cluster concentration of 7.7 mM determined by metal analysis. The relative intensities of the theoretical spectra of Fig. 6 are predicted by the theory without use of any additional parameters. We have marked in Fig. 7 the energies of the microwave quantum at X- and Q-band frequencies. It can be seen that only a small fraction, 15%, of the molecules of the sample have a A sufficiently small to permit transitions at X-band ! Figure 7 demonstrates forcefully that determination of the spin concentration of the sample is not possible without knowledge of the distribution of A, because most of the molecules do not contribute

[17]

475

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

X

Q

N,--

0

._Z, ,0 Q.

/

\ I

0

0.3

0.6

I

I

0.9 A (cm "1)

1.2

1.5

FIG. 7. Distribution of A values for the Fe3S 4 cluster of Fd II as obtained from the analysis of the spectra of Fig. 6. The vertical lines indicate the energies of the microwave quantum at X- and Q-band frequencies. Only those molecules having A values smaller than the microwave quantum at X-band contribute to the EPR spectra of Fig. 6. This graph demonstrates that one cannot obtain the spin concentration from an integer spin EPR signal without analysis of the line shapes.

to the spectrum at X-band. Note that for Fd II virtually all molecules in the sample would contribute to the spectrum if the spectrum were recorded at Q-band (-36 GHz). Finally, it is exceedingly difficult to estimate, even within a factor of 10, the spin concentration of an integer spin signal by mere inspection of the signal strength. However, from simulations of EPR spectra, a quantitative analysis of the signal can be accomplished even when the fraction of the molecules observed is only 15%, as is the case for Fd II. In many instances, the spin concentration will be known when M/Sssbauer spectra of the same sample are recorded under suitable conditions. We have stressed that the line shape of integer spin EPR signals is quite sensitive to heterogeneities, as expressed by distributions of the ZFS parameters D and E/D. The Mrssbauer spectra reflect these heterogeneities as well. The dashed lines in Fig. 3 were computed for E/D values of Fd II which correspond to the standard deviation of the Gaussian that fits the EPR spectra of Fig. 6. It can be seen that (Sz), and therefore Hint , is quite sensitive to variations in E/D during the rising portion of the curves. Because of the variations in Hi,t the MOssbauer spectra broaden.

476

PROBES I

I

-6

I

I

I

I

I

-4

OF I

METAL I

I

I

I

l

l

I

ION

ENVIRONMENTS

l

l

1

l

I

[17]

l

I

I

-2 0 2 VELOCITY (mm/sec)

I

4

I

I

6

FIG. 8. Theoretical spectra of site I of reduced D. gigas Fd II at 0.15 T and 1.5 K assuming E/D = 0.23 --+ O'E/D. The solid line was computed assuming trE/D = 0 while the simulation represented by the crosses is a convolution of spectra with a Gaussian distribution assuming

OrE/D ----- 0 . 0 5 5 .

Figure 8 shows two 1.5 K spectra c o m p u t e d with the p a r a m e t e r s of Fd II; for clarity we h a v e s h o w n only the s p e c t r u m of site I. The s p e c t r u m r e p r e s e n t e d b y the solid line was c o m p u t e d under the a s s u m p t i o n that O'E/D = 0, w h e r e a s for the c o m p u t a t i o n r e p r e s e n t e d by the crosses we h a v e c o n v o l u t e d spectra along a Gaussian distribution with O-E/D = 0.055 as d e t e r m i n e d b y EPR. (To test w h e t h e r O-E/D can be extracted f r o m the M f s s b a u e r spectra we h a v e added s o m e r a n d o m noise to the s p e c t r u m a n d a s k e d one of our first-year graduate students to extract O'E/o. H e estimated O'E/D as 0.05.) Spectra in Limit A = 0 W e h a v e r e p o r t e d a c o m b i n e d M 6 s s b a u e r and E P R study for the state pox of the P clusters o f nitrogenase that describes an integer spin s y s t e m for which the lowest two levels are separated in energy b y A < 0.01 c m - 1.7 W e found that A -< 0.001 c m - l for the P clusters of the proteins f r o m Azotobacter vinelandii, Kiebsiella pneumoniae, and Clostridium pasteurianum, and that A ~ 0.01 c m - 1for the Xanthobacter autotrophicus protein. Figure 2A shows a parallel m o d e s p e c t r u m o f the X. autotrophicus protein. N o t e the sharpness o f the r e s o n a n c e line. This sharpness is a characteristic feature of spectra for which flH ,> A; under these conditions the spectra are quite insensitive to distributions in A, and the transition probability

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

477

peaks sharply at g~ff = hv/flH with an intensity proportional to (A/fill) z. Thus, the signal vanishes in the limit A -- 0. (For the S = 2 system discussed above, A = 0 occurs for E -- 0. For E = 0 the two levels have magnetic quantum numbers M = + 2 and M = - 2 when the magnetic field is applied along z, and EPR transitions are forbidden because AM = --_4.) For many years we thought that A values as small as 0.0l cm-~ would be unlikely for metal complexes of biological interest. This is still true for the high-spin ferrous system. H o w e v e r , more recent studies in various laboratories have dealt with metal clusters having S > 2. We have pointed o u t 7 that a multiplet with spin S describable by Eq. (l) has at least one pair of levels for which A ~ IDI(E/D) s. Because E/D is confined to 0 -< E/D <- ½, small A values are likely to be observed for multiplets with large S. Consider, for instance, an exchange-coupled pair of high-spin (S = ~) Fe 3÷ sites. The exchange coupling produces a series of multiplets with S -- 0, l, 2, 3, 4, and 5, where S is the dimer spin. For the iron sites of Fe2S 2 clusters and the clusters of iron-oxo proteins typical D values are about 1 cm-~. It is thus very likely that some of the states with S -> 2 have a pair of levels with a small A. For example, we have observed a sharp resonance at geff = 8.0 for the excited S = 2 multiplet of the Fe 3+ - F e 3÷ cluster of the hydroxylase component of methane monooxygenaseH; for the transition observed we found that A ~ 0.03 cm-~. (The multiplets with S > 3 are too high in energy to be populated at temperatures when the spin-lattice relaxation is slow enough to permit observation of an E P R signal.) The P clusters of nitrogenase have been quite a challenge for the spectroscopist. For nearly 15 years it was thought that a P cluster contains four Fe sites, and that the P cluster state pox had half-integral electronic spin, S >- ~. For A _< 0.001 cm -~ the two nearly degenerate levels of pox behave essentially like a Kramers doublet, and it is therefore understandable that it took many years before the nature of the electronic state was recognized. The distinction between a Kramers doublet of a system with half-integral electronic spin and a pair of nearly degenerate levels of a non-Kramers system may appear esoteric to a biochemist. H o w e v e r , the structural implications for the two cases differ substantially. If pox were a Kramers system, spectroscopic and redox data would suggest that nitrogenase contains four Fe4S4-type P clusters. The recognition of pox as a non-Kramers system implies that each P cluster contains eight Fe sites IIB. G. Fox, M. P. Hendrich, K. K. Surerus, K. K. Andersson, W. A. Froland, J. D. Lipscomb, and E. Mfinck, J. Am. Chem. Soc. 115, 3688 (1993).

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PROBES OF METAL ION ENVIRONMENTS

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and that the protein contains two such superclusters. The latter view is supported by X-ray crystallographic studies.~2 Concluding Remarks Integer spin EPR will become an important tool for the studies of the active sites of iron-containing metalloproteins. In our laboratories we have focused on combined EPR and M6ssbauer studies of systems yielding M6ssbauer spectra that could be analyzed with high accuracy and confidence in the framework of the spin Hamiltonian formalism. Using zerofield splitting parameters from the M6ssbauer analysis, we have been able to assign EPR transitions with confidence and have refined the methods of EPR data analysis (see, e.g., Ref. 8). Integer spin EPR, in turn, has aided us in recognizing the fundamental features of some systems; in particular, it has helped us to solve the problem of the P cluster state pox. We now have sufficient experience in analyzing integer spin EPR signals that we can venture into studying noniron systems. It may be useful here to list some of the iron-containing proteins and model complexes for which integer spin EPR has been observed. The M6ssbauer spectra of the S -- 2 states of reduced Fe3S4 clusters are quite similar, and, not surprisingly, most clusters exhibit an EPR signal in this state. For those Fe3S4 proteins for which an EPR signal has not been observed at X-band, Q-band EPR is most likely to yield positive results. Integer spin EPR has been reported for a diverse class of proteins, including deoxymyoglobin as prepared or after flash-off of CO 4 or O2,13 beef heart 4,14 and yeast 4 cytochrome-c oxidase, nitrogenase, 7 iron-oxo proreins, 5'6'1~ desulfoferredoxin, ~5 and Fe(II)-substituted alcohol dehydrogenase.~6 Integer spin EPR has also been reported for model complexes of rubredoxin ~7 and iron-oxo proteins. TM One distinction should be made regarding the EPR properties of the listed proteins. The signals observed for myoglobin, 4 the oxidized hydroxylase of methane monooxygenase, ~ and K. pneumoniae and A. vinelandii nitrogenase 7 originate from transit2 j.

Kim and D. C. Rees, Science 257, 1677 (1992). 13 M. P. Hendrich and P. G. Debrunner, J. Magn. Resort. 78, 133 (1988). 14 W. R. Hagen, Biochim. Biophys. Acta 708, 82 (1982). 15 I. Moura, P. Tavares, J. J. G. Moura, N. Ravi, B. H. Huynh, M.-Y. Liu, and J. LeGall, J. Biol. Chem. 265, 21596 (1990). 16 E. Bill, C. Haas, X.-Q. Ding, W. Maret, H. Winkler, A. X. Trautwein, and M. Zeppezauer, Eur. J. Biochem. 180, 111 (1989). 17 M. T. Werth, D. M. Kurtz, Jr., B. D. Howes, and B. H. Huynh, lnorg. Chem. 28, 1357 (1989). ~8A. S. Borovik, M. P. Hendrich, T. R. Holman, E. Miinck, V. Papaefthymiou, and L. Que, Jr., J. Am. Chem. Soc. 112, 6031 (1990).

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VOLTAMMETRY OF REDOX-ACTIVE CENTERS

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tions between excited spin levels. These signals are not easily correlated with the corresponding M6ssbauer spectrum because the electronic spin has generally intermediate relaxation rates at temperatures (10-20 K) where the excited spin levels are appreciably populated; for intermediate relaxation rates the M6ssbauer spectra are broad and ill-resolved. M6ssbauer spectra are observed in the slow fluctuation limit if the electronic spin relaxes with a rate slower than 106/sec. Thus, if one observes a M6ssbauer spectrum in the slow fluctuation limit, one can be assured that EPR spectra will be observed in the slow fluctuation limit as well. A survey of the literature on M6ssbauer spectroscopy suggests that a variety of other iron-containing proteins are likely to exhibit integer spin EPR at either X- or Q-band frequencies. Based on the progress that has been made in the last few years we anticipate that this technique will be a valuable tool for the biochemist and biophysicist. By applying M6ssbauer and EPR spectroscopy to proteins with multiple iron-containing sites and using the methodology discussed above, one should be able to untangle quite complex situations. Acknowledgments The work described here was supportedby grants fromthe NationalInstitutesof Health (GM-22701) and the National Science Foundation(MCB-9096231).

[18] V o l t a m m e t r i c S t u d i e s o f R e d o x - A c t i v e C e n t e r s in M e t a l l o p r o t e i n s A d s o r b e d o n E l e c t r o d e s

By FRASER A. ARMSTRONG, JULEA N. BUTT, and ARTUR SUCHETA Introduction In recent years it has been shown that redox proteins can be induced to interact directly with an electrode surface and display reversible electrochemistry in the same way as many smaller molecules. ~,2 This has suggested the possibility of using dynamic electrochemical methods such as cyclic voltammetry to examine the many intricate functional properties of redox-active centers in proteins. In this chapter we describe a particular strategy, thus far demonstrated to be applicable for investigating labile I F. A. Armstrong, Struct, Bonding (Berlin) 72, 137 (1990). 2 A. M. Bond and H. A. O. Hill, in "Metal Ions in Biological S y s t e m s " (H. Sigel and A. Sigel, eds.), Dekker, N e w York, 1991.

METHODS IN ENZYMOLOGY,VOL. 227

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