THE JOURNAL 0~ BIO~GICA~ CHEMISTRY Vol. 265, No. 7, Issue of March 5, I+ 388@3@8E$ 1990 @ 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Miksbauer Study of CO Dehydrogenase from CZostridium thermoaceticum*

(Received for publication, June 23, 1989)

Paul A. Lindahlgs, Stephen W. Ragsdalell, and Eckard Mtinck$ From the *Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392 and the IIDepartment of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201

We have studied with Mijssbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials >-200 mV, all of the -12 irons reside in diamagnetic environments and con- tribute a quadrupole doublet characteristic of [Fe&]‘+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe&J’+ cluster. We have also observed the Mtissbauer spectrum (-18% of Fe) of the complex which yields EPR with g = 2.01, lB1, and 1.65. Also present is a doublet (9% of Fe) with AEQ = 2.90 mm/s and 6 = 0.70 mm/s, values typical of a ferrous Fe$ complex. This component seems to interact with a nickel site to form an EPR-silent complex with half- integral electronic spin. We have also characterized the iron environments of the ,S’ = Yz, NiFeC complex. This complex contributes -20% of the total MGssbauer absorption when the EPR signal has -0.35 spins/l2 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange- coupled to a [Fe&&]‘+ cluster, Finally, the Mijssbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically hetero- geneous, thus rendering the development of a model of the cluster types and compositions in this enzyme pre- mature.

CO dehydrogenase (CODH)’ from Clostridium thermouce- ticum catalyzes the reversible oxidation of CO to CO2 and the final steps in the synthesis of acetyl-coenzyme A (l-3). The enzyme has an (a& hexameric structure, and each c& dimer contains two nickel and -12 iron ions (4-6). The metals are arranged into a variety of complexes the structures of which are under investigation (7-10).

In the preceding paper (10) we reported our results of an EPR and controlled potential coulometric study of CODH. At least seven EPR signals can be observed under specified conditions. These include signals at g values of 2.08 and 2.02

* This work was supported by National Institutes of Health Grant. GM22701 (to E. M.), National Institutes of Health Grant GM10580 (to P. A. L.), Departkent of Energy Grant DE-FGO2-88ERl3875 (to S. W. R.). and a Shaw Scholars Award (to S. W. R.). The costs of publicatiok of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Present address: Dept. of Chemistry, Texas A&M University, College Station, TX 77843.

’ The abbreviations used are: CODH, CO dehydrogenase; FC, fer- rous compone&.

(termed the NiFeC signal), 2.04, 1.94, and 1.90 (thegave = 1.94 signals), 2.01, 1.81, and 1.65 (the gave = 1.82 signal), 1.97, 1.87, and 1.75 (the gave = 1.86 signal), and signals between g = 6 and 4 (the low field features). The oxidized enzyme is EPR silent. When the enzyme is reduced under an argon atmos- phere, the gave = 1.82 signal is the first to develop as the potential is lowered (E,,, = -220 mV), followed by the two gave = 1.94 signals (average E,,, z -440 mV). The NiFeC signal develops when the sample is reduced by CO or when reduced electrochemically (at potentials below -430 mV) in the pres- ence of COZ. The gave = 1.86 signal is present under these conditions as well.

Overall, these signals are presently quite difficult to under- stand. The large number present makes it difficult to study each individually, and their redox behaviors are complicated and not entirely reproducible. But the most puzzling aspect of these signals is that none yields the expected value of one spin/dimer when their intensities are quantitated.

With full awareness of these difficulties, we have under- taken a MGssbauer spectroscopic study of the enzyme. M&s- bauer spectroscopy is well suited to structurally characterize iron complexes in proteins because all of the irons in the protein can be observed, independent of their oxidation or spin state (11, 12). Moreover, when MGssbauer and EPR spectroscopies are combined, a more in-depth analysis is possible. This combination has been successfully used to identify the structures and magnetic characteristics of iron- containing clusters in a variety of proteins. In light of the complexities associated with the EPR spectra of this enzyme, our aims and achievements for this study were quite limited. In order to study the MGssbauer contributions from specific complexes, we used the electrochemical methods and the results of the preceding paper to poise 57Fe-enriched samples at various potentials.

In this paper, we report the results of this investigation. As expected, the M&sbauer spectra are extremely complex. We have been able to identify and characterize in a limited way some features of the spectra. In certain instances Mtissbauer components and EPR signals could be correlated. We have not been able to identify the reasons for the low spin quanti- tations but conclude that the enzyme preparations are spec- troscopically heterogeneous.

EXPERIMENTAL PROCEDURES

purificution-The procedures used to prepare “Fe-enriched C/OS- tridium thermoaceticum and purify and characterize CO dehydrogen- ase have been described (4, 13). Five preparations were used, with specific activities of 50 (for a partially purified sample), 200, 250,260, and 300 units/mg in the oxidation of CO to CO*. The preparations - contained between 2.0-2.2 Ni/dimer and 11.6-13.0 Fe/dimer, deter- mined according to published methods (10, 14-16).

Electrochemical Cell-The cell used to poise samples electrochem- ically (Fig. l), consists of a plexiglass top (D) (8 x 7 X 4.5 inches)

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Mtissbauer Study of CO Dehydrogenase

FIG. 1. Electrochemical cell for Mtissbauer samples. The cell is described in the text.

and an aluminum base (H) (8 x 7 x 3.5 inches). At the bottom of the base is a rotary feedthrough (G) (Varian, model 1371), througb which a % inch diameter rod (J) extends. A Mossbauer cup (l-cm diameter, l-cm height) fits tightly in a conically shaped copper holder (I?) at the top of the rod. The electrodes (I? and C) are secured into the top of the box via lo/30 outer glass joints, angled so that the electrode tips extend into the Mossbauer cup. Samples are introduced through a stopcock (A) at the top of the cell. Solutions are stirred by spinning the rod assembly at 200 rpm with a variable speed motor (Masterflex drive, Cole-Palmer) situated beneath the cell. After an experiment, the copper holder and sample are lowered into the recess of a liquid nitrogen-cooled copper block (F). The liquid nitrogen enters the block through a cryogenic feedthrough (G) (model FLH-N25X2, Kurt J. Lesker Co.) attached to the side of the base. Samples typically freeze in 2.5 min. The nylon Mossbauer cups which fit tightly in the copper holder at room temperature loosen at cryogenic temperatures, allow- ing easy removal.

In a typical experiment the cell was rendered anaerobic by vacuum pumping (with the working and dry auxiliary electrodes in place), and backfilling with oxygen-free (~0.5 ppm) argon. The auxiliary electrode was activated by the addition of a saturated KC1 solution, and the reference electrode was then inserted. The Mossbauer cup was filled with 300-450 ~1 of a buffer solution (mediator mix) con- taining -60 gM each of 5,5’-dimethyl-l,l’-trimethylene-2,2’-bipyri- dyl (Em = -664 mV), methyl viologen (Em = -440 mV), benzyl vioiogen (Em = -348 mV), Neutral Red (Em = -325 mV), Phenosaf- ranine (Em = -252 mV), Indigo Carmine (Em = -125 mV), 2,5- dihydroxy-o-benzoquinone (Em = -60 mV), methylene blue (Em = +37 mV), 50 mM potassium phosphate, pH 7.6, and 0.1 M KCl. A 2. h exposure of the mediator mix at a controlled potential (CV27 potentiostat, Bioanalytical Systems) while under a constant flow of argon was required to obtain a constant background current (O-4 PA). Between 25 and 300 ~1 of a thionin-oxidized protein sample was then introduced, and the additional current due to the reduction of the

protein was recorded. Protein samples were prepared as described (10). The current returned to background typically within 1 h. Am- perometric data was collected on an IBM-AT computer using a Data Translation acquisition board (DT2800) and a program (ELECTRO) written in the ASYSI@ (McMillian Co.) language. Backgrounds sub- tracted from the data were polynomials of first, second, or third order, generated from the data collected prior to the injection of the sample, and from that collected after the reduction was complete.

The working electrode (Fig. 1B) was constructed from four gold helical wires housed in a modified inner lo/30 ground glass joint. The Ag/AgCl auxiliary electrode (Fig. 1C) was constructed in similar fashion, with a small stopcock attached to the top of the joint and a piece of thirsty glass (Corning) attached at the bottom with heat- shrink tubing. The saturated calomel reference electrode was also constructed from a ground glass joint, with a second glass tube (parallel to the main joint, but displaced laterally) attached at the top of the joint, and a glass frit (Fisher No. 13-641-598) attached at the bottom. A Pt wire was inserted through the bottom of the second glass tube so as to contact the Hg on the inside of tbe tube. Controlled potential coulometric reductive titrations of oxidized methyl viologen were used to calibrate the reference electrode. For all seven such titrations performed, the data lit well to the Nernst equation where n (number of electrons transferred/redox site) equaled one. The reductions quantitated to an average of 0.95 ? 0.07 mol of electrons/ mol of viologen. An average midpoint potential of E,,, = -690 k 10 mV uersm saturated calomel reference electrode was obtained. Using an E,,, = -440 mV uersus NHE for methyl viologen (17), we obtained a value of +250 mV uersus NHE as the potential of the reference electrode. Solution potentials are quoted uerws NHE at pH -7.2.

The Mossbauer facilities have been described (18). EPR spectra were obtained on a Varian El09 spectrometer fitted with an Oxford ESR910 cryostat. EPR spectra were manipulated using a program (EPRDAT) written in ASYST. Quantitations were performed using a 0.217 mM CuEDTA standard as described (19).

RESULTS

Before presenting the M&sbauer spectra of CODH, a few remarks about the genera1 features of Mossbauer spectra may be useful. For a comprehensive treatment the reader may consult Refs. 11, 20, and 21. The Mossbauer spectra of Kra- mers systems (complexes with half-integral electronic spin) and non-Kramers systems (integer or zero electronic spin) differ in a fundamental way. In zero applied magnetic field, the Mossbauer spectra of non-Kramers systems do not exhibit paramagnetic hyperfine structure (magnetic splittings); each iron site yields only a quadrupole doublet. By applying an external magnetic field one can assess whether the non- Kramers system is diamagnetic (S = 0) or paramagnetic (S zz 1 integer). In contrast, Kramers systems exhibit in general, at sufficiently low temperatures (typically at 4.2 K), paramag- netic hyperfine structure, even in zero applied magnetic field. Since Kramers systems are in general EPR active, these facts allow us to correlate the Mossbauer and EPR spectra of EPR active clusters. The magnetic features of Mossbauer spectra are controlled by the so-called internal magnetic field, IYint = -A. (S)/g,& where A is the magnetic hyperfine tensor and (S) is a suitably taken expectation value of the electronic spin S. At low temperature, in the limit of slow spin relaxa- tion, (S) is simply the expectation value taken for the two members of the lowest Kramers doublet; for an isotropic S = % system, (SZ) = ? r/z, where z refers to the direction of the applied field. At higher temperatures (typically at T > 100 K), the electronic spin fluctuates in general fast and (S) is replaced by a thermal average, ( S)th. Since the thermal av- erage contains positive and negative (SZ) values in almost equal weights, (SZ)th - 0, and the magnetic hyperfine inter- actions average out. Under these conditions one observes quadrupole doublets for Kramers systems.

In the following we present a variety of Mossbauer spectra, starting with the most oxidized state and continuing with the more reduced states of CODH. This allows us to correlate

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3882 M&sbauer Study of CO Dehydrogenase

Mkbauer spectra with features observed by EPR. Table I summarizes the components observed in spectra of reduced CODH.

OG&ed CODH-Thionin-oxidized CODH was electro- chemically poised at +450 mV. M5ssbauer spectra of this material, recorded at 4.2 K in parallel applied fields, are shown in Fig. 2. The spectrum of Fig. 2A, obtained in a field of 600 gauss, consists essentially of one quadrupole doublet with quadrupole splitting, A&, = 1.1 mm/s, and isomer shift, 6 = 0.44 mm/s. Such parameters are typical of Fe.& clusters in the 2+ core oxidation state. The solid line drawn through the data of Fig. 2A is a doublet with linewidth 0.38 mm/s. This width is somewhat larger than the typical width of 0.27-0.30 mm/s, and it reflects, of course, the fact that approximately 12 iron environments contribute. The spectrum in Fig. 2B was recorded in an applied field of 6.0 T. The solid line is a theoretical spectrum generated with the assumption that all iron sites are diamagnetic (S = 0). The good fit to the splitting pattern supports this assumption. Another sample, poised at 0 mV, exhibited essentially the same spectra. This suggests that all iron-containing complexes in CODH remain in their

TABLE I Summary of identified CODH spectrul components

Component

&! a”e = 1.82 FCII FCI NiFeC

(0x1 WI

A& 6 Total absorption

mm/s %

-0.9 -0.4 -20 2.9 0.7 9 2.2 0.61 19-20

1.15 0.44 18 0.9-1.5 0.42

I , , , , , , , , , , -4 -2 0 2 4

vELocrrY (mm/s)

FIG. 2. Mtissbauer spectra of CODH poised at +450 mV. A, 4.2 K spectrum recorded in a field of 60 mT applied parallel to the observed y-radiation. B, 4.2 K spectrum recorded in a 6.0 2’ parallel field. The solid lines are simulated spectra assuming one site with AEQ = +l.lO mm/s, 7 = 0.5, and 8 = 0.44 mm/s. For the simulation of the 6.0 T spectrum we have assumed S = 0 for all sites.

oxidated, diamagnetic forms at potentials between 0 and +450 mV.

The goue = 1.82 Complex-Upon lowering the potential, CODH samples develop an EPR signal with g values at 2.01, 1.81, and 1.65. This species, which we call the gave = 1.82 species, develops with a midpoint potential of = -220 mV (10). At -300 mV potentials, it is essentially the only EPR signal present in our samples. In order to study the gave = 1.82 species with Mksbauer spectroscopy, we have prepared two samples. One was poised at -300 mV, a potential sufficient to reduce 90% of the gave = 1.82 complex. Another was poised at 0 mV. The EPR spectrum of the -300 mV sample exhibits the gave = 1.82 signal (Fig. 3, lower panel) with an intensity corresponding to 0.2 spins/l2 Fe; this spin concentration is typical for a fully reduced sample. The MGssbauer spectrum of the -300 mV sample (Fig. 3A) exhibits a paramagnetic component which contributes shallow absorption between -2.5 mm/s and +4 mm/s Doppler velocity. This paramagnetic component, absent in the 0 mV sample, corresponds to roughly 20% of the total iron in the sample. Since the com- ponent exhibits magnetic hyperfine interactions even in zero field, it must belong to the complex giving rise to the gave = 1.82 signal.

-300 rnV, 4.2 K

'; ;Y, // , ,

', I'

3250 3450 3650 3650 4050 II (gauss)

FIG. 3. Miksbauer and EPR spectra of CODH poised at -300 mV. A, 4.2 K spectrum recorded in a 60 mT parallel field. B, 135 K M%sbauer spectrum recorded in a 60 mT parallel field. C, 10 K EPR spectrum of CODH poised at -300 mV. EPR conditions were microwave power, 50 microwatts; microwave frequency, 9.228 GHz; modulation amplitude, 10 G. D, 10 K EPR spectrum of CODH poised at 0 mV. EPR conditions were as in C. Receiver gain and protein concentration for C and D were the same, and the spectral intensities may therefore be directly compared.

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Mksbauer Study of CO Dehydrogenase 3883

At temperatures above 40 K the electron spin of this com- plex fluctuates fast on the time scale of Mossbauer spectros- copy (-10e7 s). Under these conditions, the magnetic hyper- fine interactions average out, and the complex contributes only quadrupole doublets. Inspection of the T = 135 K spec- trum of Fig. 3B shows that these doublets are masked by the main doublet of the diamagnetic material. This prevents us from determining A& and L? reliably. Since the quadrupole splittings of the species which contribute to the main doublet are essentially independent of temperature, we can estimate A& and 6 from a difference spectrum (4.2 K minus 135 K) and obtain Al& - 0.9 mm/s, and IJ - 0.4 mm/s.

Ferrous Component II-We have observed in various re- duced samples at temperatures above 50 K, a quadrupole doublet with A& = 2.9 mm/s, and 6 = 0.7 mm/s. This doublet quite consistently represents -9% of the total iron. The quoted parameters are typical of those observed for high-spin ferrous ions in a tetrahedral environment of thiolate ligands. We refer to this species as ferrous component II (FCII). The high energy line of FCII appeared at a Doppler velocity of +2.2 mm/s in 90 K spectra of samples which were 1) poised at -350 mV, 2) poised at -523 mV (Fig. 4D), 3) reduced by CO (Fig. 6B), or 4) reduced by dithionite. The low energy line of FCII appeared at -0.7 mm/s (see Figs. 4C and 6B).

Interestingly, FCII does not appear as a quadrupole doublet in 4.2 K spectra, even in those spectra obtained in zero applied

2 %O

iI

3 2

0

I

, , , , , , I , , , 1

-4 -2 0 2 4 VELOCITY (mm/s)

FIG. 4. 60 mT parallel field Mtissbauer spectra of CODH poised at -358 or -523 mV. A, -358 mV sample at 4.2 K. B, -523 mV sample at 4.2 K. C, -358 mV sample at 9iK. D, -523 rnv sample at 90 K. The solid kne labeled I outlines the features of the qua&pole doublet FCI. In C, FCI is drawn to represent 6.4% of the total absorption area, while in D, it is drawn at 20%. The so& kne labeled II outlines component FCII. In both C and D, FCII is drawn to represent 9% of the absorption.

field. This suggests that the spectrum of FCII exhibits mag- netic hyperfine interactions. Thus, FCII belongs most likely to a complex with half-integral electronic spin. Since such complexes are, with few notable exceptions, EPR active, we have searched for EPR signals which could be associated with the complex containing FCII; we have failed to observe an appropriate feature. Our data show clearly that none of the EPR active species of CODH are associated with FCII. FCII is absent (see Fig. 3B) when the gave = 1.82 signal is present, so it could not arise from this species. FCII is present in spectra where the gave = 1.94, 1.86, and NiFeC signals are either absent or nearly absent (Fig. 4C, 6B), so it could not arise from these species either.

We are severely limited in analyzing the magnetic proper- ties of the FCII-associated complex because the magnetic Mossbauer components are unresolved, and superimposed with those of gave = 1.82 species as well as with the intense quadrupole doublet arising from the remaining diamagnetic material. However, it does appear that the FCII-associated magnetic material constitutes only 7-10% of the total spectral absorption, which is no more than that obtained by quanti- tation of the FCII doublet alone. Consequently, this complex does not appear to contain iron sites in addition to FCII.

[4Fe-4S]‘+11+ Cluster-In order to study the cluster(s) that yield the gave = 1.94-type signals we have prepared two sam- ples. One was poised at a potential of -358 mV and another at -523 mV. In the former sample the cluster(s) which give rise to the gave = 1.94 signals should be mainly in the S = 0, oxidized state; in the latter sample the cluster(s) should be in the gave = 1.94 state(s). This expectation is confirmed by the spectra of Fig. 4. The 4.2 K spectrum of the -358 mV sample (Fig. 4.4) exhibits the central doublet of Fig. 2, and magnetic components (-38% of total Fe) which contribute from about -3 mm/s to +3 mm/s Doppler velocity. A comparison of this spectrum with that of the -523 mV sample shows that for the latter sample the intensity of the central doublet has declined and that of the magnetic components has increased. The shape as well as the overall magnetic splittings of the dominant magnetic component is typical of the spectral pat- terns observed for the S = i/z forms of [Fe&]‘+ clusters. This is supported by an examination of the 90 K spectra. At 90 K the electronic spin relaxation rate of the g = 1.94 species is fast on the Mossbauer time scale, and, consequently, the clusters which give rise to the g = 1.94 signals should contrib- ute only quadrupole doublets at 90 K. The most noticeable difference between the spectra of Fig. 4C and D is a quadrupole doublet labeled ferrous component I (FCI). Its Mossbauer parameters are A& = 2.2 mm/s and 6 = 0.61 mm/s at 90 K, and it accounts for 19-20% of the total iron in the sample. This doublet belongs almost certainly to S = i/z, g = 1,94- yielding, [Fe&]‘+ type clusters. The evidence for this is as follows.

Formally, [Fe&]‘+ clusters contain one ferric and three ferrous ions. However, with Mossbauer spectroscopy these sites are observed as two pairs of equivalent ions, namely a [Fe’+Fe*+] ferrous pair and a delocalized [Fez+Fe3+] pair (22). The iron sites of the ferrous pair have positive magnetic hyperfine couplings, and their AEe and 6 values are in the range 1.5-2 mm/s and 0.58-0.62 mm/s, respectively. The [Fe*+Fe3+] pair, on the other hand, has negative coupling constants and smaller AZ& and 6 values; L?Q - 0.7 - 1.3 mm/ s and 6 - 0.50 - 0.54 mm/s. Although the A,!& of FCI is slightly larger than the AJ!& values observed for other systems, we have little doubt that this component belongs to ferrous pairs of [Fe&]i+ clusters. Since the A& and ?j parameters of the [Fe*+Fe3+] pair are generally smaller than those of the

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3i384 Miissbauer Study of CO Dehydrogenase

ferrous pair, we expect that the [Fe2+Fe3+] pair contributes at 90 K a doublet that essentially overlaps the central doublet in Fig. 4D, and would thus not be resolved.

We have obtained the Mossbauer spectra of seven different samples which we attempted to reduce fully. FCI accounted for 16-20% of the total iron in the samples. This leads us to believe that FCI, when fully developed, represents -20% of the iron in CODH. Since the ferrous pair of an [Fe&]‘+ cluster represents half of its iron, -40% of the total iron in CODH should belong to fully reduced g = 1.94 type [Fe&]‘+ clusters.

Essentially all of the magnetic iron in both the -358 and -523 mV samples can now be accounted for. We observe, in the -523 mV sample, that -74% of the iron exhibits magnetic hyperfine structure at 4.2 K. The 90 K spectra show that 40% of the iron belongs to the FCI-associated [Fe&]‘+ cluster(s), 9% to FCII, and we expect 20% of the iron to be associated with the structure that yields the gave = 1.82 signal. These contributions total to 69%, within error of 74%.

The EPR spectrum of the -523 mV sample (sample of Fig. 4B and D) showed that the gN = 1.94 and gB = 1.94 signals were both present with roughly equal intensities. In addition, the EPR spectrum exhibited the gave = 1.82 signal. Thus, to which g = 1.94 cluster type do we assign FCI? Our data suggest that both the gN = 1.94 and the gB = 1.94 clusters contribute to FCI, for the following reason: FCI belongs to a cluster which accounts for 40% of the total iron in the sample. Each of the g = 1.94 signals typically represents -0.3 spins/ 12 Fe. Thus, it is reasonable to assume that each species contributes about equal absorption in the Mossbauer spec- trum. If FCI would belong only to one of the g = 1.94 species, then bothg = 1.94 species together should contribute magnetic patterns at 4.2 K accounting for approximately 80% of the total absorption. Since 30% of the magnetic material at 4.2 K belongs to the gave = 1.82 cluster and to FCII, and since the total magnetic components at 4.2 K account for 74%, the assignment of FCI to either the gN or gB cluster alone would be in conflict with the 4.2 K data.

As pointed out in the preceding paper, we found that the EPR spectra of CODH consistently exhibited resonances which are most reasonably attributed to S = 3/2 forms of Pe&ll+ clusters (10). From our earlier work on the Fe- protein of nitrogenase (23, 24), we can estimate from the shape of the spectrum of Fig. 4B that the S = 312 form, if it has a shape similar to the Fe protein cluster, must represent less than 10% of the total Fe; otherwise the troughs at +2 mm/s and -1 mm/s Doppler velocity would be “filled” by the contribution of the S = 3/2 form. Furthermore, the high temperature data indicate that S = 3,/2 clusters are not associated with FCI: the extent of valence delocalization in S = 3/2 clusters is greater than in the corresponding S = l/2 clusters (23-25). Consequently, in S = 3/2 clusters, all four iron sites have approximately the same, intermediate, Al& and fi values and contribute a single, broadened quadrupole doublet at high temperatures. This is clearly not the case for FCI; it is distinct and well isolated from its [Fe3+Fe2+] partner.

To summarize, the Mossbauer data show that at least 40% of the total iron in the reduced sample belongs to S = l/z [Fe&,]‘+ clusters. The clusters occur in two forms. Both forms contribute to FCI and both have the same g values. However, the two cluster forms yield EPR resonances of different width and different relaxation behavior. EPR suggests that some iron is in the form of S = 3/2 [Fe&]‘+ clusters. The Moss- bauer data indicate that this species is present in minor amounts (-lo%), and is associated with some species besides the FCI [Fe4SJ’+ cluster(s).

~&C CLrter-When CODH samples are reduced with CO, or by low potential mediators in the presence of CO*, an EPR signal with g1 = 2.08 and g ,, = 2.02 develops. The signal typically represents about 0.3 spins/l2 Fe. Isotopic substitu- tion experiments with “Ni, 67Fe, and 13C0 have shown that the signal belongs to a complex that contains Ni, Fe, and CO- derived carbon (8, 9). The signal can be observed at temper- atures up to 170 K. This suggests that one can study the Mossbauer spectra under conditions where the spin of the NiFeC complex relaxes slowly on the Mossbauer time scale and where all other spins, i.e. those of the g = 1.94 and 1.82 species, relax fast. Under such conditions, the spectra of the iron sites associated with the NiFeC complex will exhibit magnetic hyperfine structure whereas all other paramagnetic centers will contribute quadrupole doublets.

We have reduced many Mossbauer samples using CO, and report on two such samples here. Both exhibited the g = 1.94 and 1.86 signals with near equal signal amplitudes. The main difference between the samples was in the strength of the NiFeC signal.* One sample exhibited this signal at a spin concentration of 0.35 spins/l2 Fe; the Mossbauer spectra are shown in Figs. 5A and 6A. The other sample had 0.1 spins/l2 Fe for this signal, and its spectra of both samples show the doublets of FCI and FCII at 19 and 9% of total Fe, respec- tively. This indicates that the structures which yield FCI and FCII are distinct from that contributing the NiFeC signal. since the EPR spectra of both samples contain the gN = 1.94, ga = I.94 and I.86 signals with about equal concentrations, and since the Mossbauer spectra show equal amounts of FCI and FCII in both samples, the combined data suggest that the contributions of these three species will cancel when the 4.2 K Mossbauer spectra of the two samples are subtracted from each other. The difference spectrum thus obtained shows a paramagnetic component, superimposed with an “inverted” quadrupole doublet with A& = I.15 mm/s and 6 = 0.44 mm/ s; the quadrupole doublet is the conspicuous feature in the center of Fig. 5B. By subtracting this doublet from the differ- ence spectrum, the spectrum of Fig. 5C was obtained, the latter should be a reasonable representation of the spectral contribution of the NiFeC complex. The spectrum shown in Fig. 6A was recorded at 90 K for the sample which yielded 0.35 spins/l2 Fe. It can be seen that at Doppler velocities < -1 mm/s and > +2 mm/s the spectrum contains a paramag- netic component which is essentially absent in the 0.1 spins/ 12 Fe sample of Fig. 6B. The outermost absorption features and the overall intensity of this paramagnetic component correspond well with the spectrum of Fig. 5C.

In order to assess the hyperfine parameters of the NiFeC complex, we have performed spectral simulations in the framework of an S = s spin Hamiltonian. Although the limited spectral resolution and the uncertainties introduced

* Our intention was to produce maximum NiFeC signal intensities with both samples. As it happened, the sample yielding the intense NiFeC signal was purified somewhat differently than the other sam- ple, in that no gel filtration step was performed. This may be of some significance because it has very recently been proposed that the CODH holoenzyme is an cz&-rz hexamer rather than an (q!3)3 hex- amer, and that the (metal-free) 7 subunit is lost during the gel filtration step (26). The sample purified without the gel filtration step contained the 7 subunit and had a specific activity of 50 units/ mg protein. Whether inclusion of the 7 subunit caused this sample to exhibit a high spin concentration of the NiFeC signal remains to be clarified. We have produced strong NiFeC signals with prepara- tions containing only the o and @ subunits, but we have not studied this effect quantitatively. The metal content of the a/3 dimer does not appear to be affected by the presence of the 7 subunit, since we obtained 11.6 Fe and 2.2 Ni/a@ dimer for the -r subunit-containing sample.

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Mkbauer Study of CO Dehydrogenase

0.0 - -c

0.5 - , , , , , , , , , II

-4 -2 0 2 4 VELOCITY b-an/~~

FIG. 5. 4.2 K Mhssbauer spectra of CO-reduced CODH. Spectra were recorded in a 60 mT applied parallel field for samples which gave NiFeC signal spin concentrations of 0.35 spin/l2 Fe (A) and 0.10 spin/l2 Fe (E). The brackets mark a quadrupole doublet with AEo = 1.15 mm/s, 6 = 0.44 mm/s; this doublet most likely represents the oxidized NiFe center. Shown in C is a difference spectrum ohtained by subtracting the spectrum in Z3 from that in A and by removing the remnant of the AEQ = 1.15 mm/s doublet. The solid line in C is a spectral simulation with an S = l/z spin Hamiltonian assuming two sites with the same AEo and 8 values, AEQ = 1.15 mm/ s and 8 = 0.44 mm/s, but distinct magnetic hyperfine tensors; Ax = Ay = 33.5 MHz and AZ = 29.5 MHz for site 1 and Ax = 33.5 MHz and A? = AZ = 25 MHz for site 2. For asymmetry parameters we used r~ = 0.5 for both sites.

by the spectral manipulations preclude a detailed analysis, some reliable conclusions can be drawn from such simulations. First, about 18% of the iron in the 0.35 spins/l2 Fe sample belong to the EPR-active state of the NiFeC complex. Second, simulations suggest that the spectrum of Fig. 5C contains two slightly distinct iron sites; the parameters used are quoted in the caption of Fig. 5. The following features of this parameter set seem significant to us: the set provides a spectral simula- tion which traces the data of Fig. 5C quite well and provides a good estimate for intensity (16% of total Fe) and the average isomer shift (&” = 0.42 mm/s). It is almost certain that the magnetic hyperfine tensors are quite anisotropic. Thus, there seems to be major components at 33.5 MHz and a minor component around 25 MHz. Since AEo is unknown for both sites there is some flexibility (&5%) in adjusting the largest A-tensor components. Adequate fits are obtained for the range of A& values between 0.9 mm/s and 1.5 mm/s.

A comparison of the spectra of Fig. 5A and B suggests that the quadrupole doublet with A& = 1.15 mm/s and 8 = 0.44 mm/s represents the iron of the NiFe complex before it has reacted with CO to form the NiFeC center. A comparison of the isomer shifts suggests that the average oxidation state of

r

, , , , , , I,, ! 1 -4 -2 0 2 4

VBLOCITY (nun/s)

FIG. 6. Miksbauer spectra of CO-reduced CODH. A, The spectrum of the sample of Fig. 5A, recorded at 90 K in a parallel 60 mT field. The solid line, drawn to represent 18% of the total ahsorp- tion, is the same curve as shown in Fig. 5C. Contributions from FCI and FCII are indicated. E, 60 mT spectrum of the sample of Fig. 5S, recorded at 135 K. The solid lines show the contributions of FCI and FCII, drawn to represent 19 and 9% of the absorption area, respec- tively.

the iron is the same in both states. We will discuss the ramifications of this below.

g Cl”e = 1.86 Complex-We have attempted to characterize the gave = 1.86 complex with Mossbauer spectroscopy. We have observed at low potentials a magnetic compound which can be attributed to this complex. Since, however, the gave = 1.86 signal is observed when the g = 1.94 signals are present, our data lack resolution, Thus, currently we can only state the gave = 1.86 signal arises from an iron-containing center.

DISCUSSION

Here and in the preceding paper we have reported EPR and Mossbauer studies of CODH samples poised over a wide range of redox potentials. What has puzzled us most throughout these studies is that the spin concentrations of all observed EPR signals are substantially below 1 spin/l2 Fe. Although there was some variability in the intensities of EPR compo- nents for samples prepared under the same conditions, the spectra were essentially reproducible; typical spin concentra- tions of each center were 0.2-0.3 spins/l2 Fe. It is well known that a variety of effects can reduce observed spin concentra- tions. These include fast spin-lattice relaxation, spin-spin interactions caused by the aggregation of molecules, and spin- dipolar or exchange interactions between closely spaced par- amagnetic centers. Our Mossbauer data show clearly that none of these effects are the cause for the low spin concentra- tions. Other phenomena, such as clusters occurring in mix- tures of different spin states such as observed for the nitro- genase Fe protein (23, 24), or molecules with partially empty sites, could also lower spin concentrations. The presence of low field EPR signals indicates that spin state mixtures are present in CODH, and our Mossbauer results, to be discussed

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3886 Mksbauer Study of CO Dehydrogenase

below, suggest that the distribution of clusters in the enzyme may be heterogeneous as well.

The Mossbauer spectra of the oxidized enzyme, taken alone, would suggest that all iron belongs to FedSa clusters in the Z+ core oxidation state. It is clear from our studies of the reduced enzyme that at least 40% of the iron belongs to such clusters. Whatever structures the remainder of the iron belongs to, they give, in the oxidized enzyme, Mossbauer signatures typ- ical of [Fe,S#+ clusters.

This study has shown that reduced CODH contains at least four types of iron-containing complexes; namely, the gave = 1.82 complex, FCII, the FCI-associated [Fe&] cluster(s), and the NiFeC complex. We will now summarize our knowledge about each of these species.

Reduced CODH contains at least one [Fe&]‘+ cluster. The cluster yields component FCI in Mossbauer spectra and two distinct g = 1.94 type signals which have the same g values but different linewidths and saturation behavior.

Since the gave = 1.82 center yields broad and unresolved low temperature Mossbauer spectra, few conclusions about the structure of this complex can be drawn. Under conditions where the quantitation of the EPR spectrum yielded -0.2 spin/l2 Fe, we observed that -20% of the iron in our sample was associated with the gave = 1.82 center, Its g values, g = (2.01, 1.81, 1.65) are reminiscent of those exhibited by binu- clear iron-oxo complexes (27). The Mossbauer spectra of the latter exhibit, at temperatures above 100 K, two quadrupole doublets typical of high spin Fe3+ and Fe’+ sites. Careful examination of the spectra of CODH did not provide any evidence for the presence of an appropriate Fe’+ component. The gave = 1.82 spectrum is also reminiscent of a signal (g = 2.01, 1.85, and 1.78) obtained when substrate is bound to the D%%ll+ cluster in aconitase (28). Finally, a comparison of our enzyme with the CODH from Rhodospirillum rubrum may be useful. The latter enzyme (which lacks the acetyl synthase activity) exhibits an EPR signal which resembles our gave = 1.82 species. Stephens et ul. (29-31) have shown that their signal A (g = 2.01, 1.88, 1.72) results from a nickel and iron- containing complex. Arguing by analogy, it is possible that the complex yielding the gave = 1.82 signal observed here contains nickel as well as iron.

Component FCII most likely originates from a cluster con- taining a single iron in a tetrahedral environment of predom- inately sulfur ligands. The monomeric site of ferrous rubre- doxin has t,he same AEo and 6 values as FCII, but unlike FCII it does not exhibit magnetic hyperfme interactions at 4.2 K in zero applied field. This behavior of FCII is more like that of a spin-coupled spin system. The Mossbauer spectra of reduced (S = i/2) [FeSz] ferredoxins exhibit a similar ferrous component at high temperatures and show paramagnetic hy- perfine structure at 4.2 K (12). However, the [Fe&SJ1+ clusters typically yield an intense g = 1.94 signal below 100 K; we have not observed an EPR signal which could be associated with FCII. The oxidized P-clusters (Pox) of nitrogenase, per- haps clusters with an [Fe&] core, exhibit a doublet like FCII at T > 70 K (18). Moreover, Pox has half-integral electronic spin and is EPR silent like FCII. However, Px yields a well- resolved magnetic pattern at 4.2 K, in contrast to FCII which seems to contribute an indistinct pattern. Since no other irons appear to be associated with FCII, its low temperature behav- ior is perhaps best explained by assuming that the spin of a Nil+ or Ni’+ site is coupled to the S = 2 spin of the FCII iron.

We have made an attempt to characterize the iron environ- ments of the cluster which contributes the NiFeC EPR signal. We have observed this cluster in two distinct states; namely, an oxidized form where all iron environments are diamagnetic

(with AEo = 1.15 mm/s and 6 = 0.44 mm/s) and a reduced state associated with a complex containing carbon derived from CO. Our data suggest that, in the absence of CO, the NiFe center remains in an oxidized state even at a potential of -523 mV; this is indicated by the presence of a quadrupole doublet (-26% of total Fe) in the spectrum of Fig. 4B. While it is clear from EPR studies that Ni and Fe reside in a single complex in the state which yields the NiFeC signal, it is presently not known whether both iron and nickel ions are components of the oxidized complex. Thus, it is possible that, upon reaction with CO, a nickel site becomes linked to an iron-containing cluster via a carbonyl bridge.

What type of iron-containing cluster is most likely present in the NiFeC complex? According to our analysis, the cluster seems to contain at least two types of iron sites. In the oxidized enzyme, the iron environments of this complex have param- eters (AEo, 6, S = 0) characteristic of clusters with [Fe&]*+ cores. In particular, the isomer shift, 6 = 0.44 mm/s, is indicative of this cluster type ([Fe&$‘+ have ?i 5 -0.30 mm/ s and AEo = 0.6-0.7 mm/s). Upon reaction with CO, the sites exhibit magnetic hyperfine interactions with A-tensor com- ponents ranging from 25 to 33.5 MHz. The spectral parame- ters observed for the reduced NiFeC complex are not typical of those reported for any known type of iron-sulfur cluster. While the magnetic hyperfine interactions have magnitudes comparable to those of [Fe$Sd13+ and [Fe.&]‘+ clusters, the isomer shifts of these iron sites (6 = 0.44 mm/s) indicate that their oxidation states have not changed upon reduction. In fact, if the iron environments would be those of Fe& clusters, the 2-t core oxidation state would be unambiguously indicated by the value of 6. We do not recognize any features in the parameter set listed in the caption of Fig. 5 which remind us of Fe&!& or Fe$$ clusters.3

These observations are compatible with the following model. The iron-nickel complex consists of an Fe& cluster linked by a bridging ligand to a nickel site. In the oxidized state of the enzyme, the cluster is in the 2+ core oxidation state (S = 0) and the nickel site is low spin Ni’+ (S = 0). Upon reaction with CO the nickel becomes reduced to Nil+ (S = l/z), with the iron-sulfur cluster remaining in the 2+ state. (While it is known from EPR that the carbon-contain- ing moiety is bound to the center, it is not necessarily bridging the nickel and the cluster.) The observed S = l/z spin would thus reflect essentially the nickel site. The magnetic hyperfine interactions observed for the [Fe&]*+ cluster could result from a mechanism similar to that described (34, 35) for the coupled siroheme-Fe& chromophone of Escherichiu coli sul- fite reductase; namely, that exchange interactions mediated by the ligand bridging the Nil+ site with the Fe.&, cube will mix paramagnetic (S = 1) excited state configurations of the cluster into the diamagnetic ground state, thus rendering the cluster paramagnetic. It was shown for sulfite reductase (34) that the magnetic hyperfine interactions observed at the iron sites of the Fe& can be written as Sh .Ai. I; where Sh = 512 is the electronic spin of the heme and Z, is the “‘Fe nuclear spin of iron-sulfur cluster site i. For the situation contemplated here a similar mechanism would give rise to terms of the form ,!&.A;.I, with SNi = Y2. We would like to stress that the magnetic hyperfine interactions observed here are five times larger than those reported for sulfite reductase. However, for

‘We have considered the possibility that the NiFe center consists of a cubane NiFe& cluster. We have successfully incorporated a variety of metals into the core of the Fe& cluster of Desuljou&io g&.s ferredoxin II (32). Two incubation attempts with Ni yielded clusters spectroscopically very distinct from the NiFe center of CODH (33).

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Miissbauer Study of CO Dehydrogenase 3887

such a spin-coupled system, the magnitude of the A values depends critically on the ligand(s) bridging the cluster with the nickel site.

For sulfite reductase the spectroscopic evidence for a cou- pled system was based on the study of samples which yielded highly resolved EPR (-1 spin/mol) and Mossbauer spectra. This allowed the authors (34) to develop compelling spectro- scopic arguments for the coupled arrangement, which has been subsequently confirmed by an x-ray crystallographic study (36). The arguments for a coupled Ni:Fe& chromo- phore in CODH are quite weak indeed and the case is, at best, a minimum working hypothesis for further inquiries. Bastian et ul. (37) have recently proposed a similar model for their EXAFS study of CODH. These authors point out that fits to the nickel EXAFS data can be improved by including a nickel- metal interaction at 3.25 A, a distance compatible with the bridging arrangement described here.

We have difficulties reconciling our Mossbauer data with the metal content of CODH, as well as with the low spin concentrations of each EPR signal observed. The prepara- tions of CODH used here contained an average of 2.0 nickels and 12.5 irons/dimer, similar to the values of 1.7-2.1 nickels (4, 37, 38), and 10-11 (4, 37) irons/dimer reported previously. The proportion of the Mossbauer absorption contributed by each center should reflect a multiple of the number of irons in each species (provided that each center in every protein molecule is present in only one oxidation state), the total of which in the case of CODH should sum to cu. 10 - 13 irons. The observed ratios for each component in the spectra were:

Complex: FCI-[Fe&] : NiFeC : geve= 1.82 : FCII : Unassigned Ratio: 4 :z : 2 :1 : 1

One might conclude that each CODH dimer contains one of each type of cluster and that the absorption ratios are equal to the number of irons in each center. Accordingly, in each CODH molecule there would be a [Fe&] cluster, two 2Fe- containing clusters (NiFeC and gave = 1.82), and a cluster containing a single iron (FCII). This assumption appears to be justified for the [Fe&,] cluster and the FCII species. The problem is that the irons of the NiFeC and gave = 1.82 complexes do not have characteristics of known 2Fe com- plexes. Those in the NiFeC species in particular appear most characteristic of Fe& clusters. Because of this, and because the spin concentrations of all EPR-active centers (with the possible exception of the [Fe&]i+ cluster) are substantially below 1 spin/dimer, we are compelled to conclude that our CODH samples are heterogeneous in the distribution of clus- ters. It seems unreasonable to assume that the current prep- arations could be manipulated into a state where, for instance, the gave = 1.82 signal would represent 1 spin/l2 Fe. Since we observe that 0.2 spin/l2 Fe represent 20% of the total Fe, the achievement of 1 spin/l2 Fe would then represent all of the iron in the sample, a result which would conflict with our data. Thus, our data suggest that a substantial fraction of the molecules lacks the structures which yield the gave = 1.82 signal. A similar argument can be applied to rationalize the low spin concentration observed for the NiFeC center. These considerations, of course, imply that the total number of irons/molecule in the holoprotein is substantially higher than current estimates.

This heterogeneity does not necessarily imply that some sites are occupied with cluster fragments, although this pos- sibility cannot be excluded. In many respects, the clusters of CODH are quite stable as witnessed by the fact that potential changes of as much as 700 mV are reversible, without any evidence of cluster destruction. Moreover, the heterogeneities

are not apparent in the chromatographic behavior of the protein.

The proposed spectroscopic heterogeneity may also provide a plausible explanation for the differences in the temperature and saturation properties of the gN = 1.94 and gs = 1.94 signals; the former signal may arise from molecules with one distribution and the latter signal from molecules with another. The proportion containing the gB = L94-yielding [Fe&]*+ cluster may have a neighboring paramagnet enhancing its electronic spin relaxation rate, while those containing the gN = 1.94 species may not. Our data do not allow us to decide whether the ,gN and gB clusters are variants of the same cluster at one site or whether they are two distinct clusters located in different sites (with every other site filled). However, since component FCI has quite sharp lines, and since its A& value is quite distinct, we consider the former possibility more likely. If the clusters would reside in different sites, the local environments would have to be remarkably similar in order to produce exactly the same values for A& and 6 of doublet FCI.

Clearly, more work is required to elucidate the nature of the clusters in CODH. Among the most crucial problems which must be solved are the causes of the low spin quanti- tations, and the achievement of spectroscopically homogene- ous protein preparations. The solution to these problems may require a means of altering the electronic state of the clusters in CODH, possibly through the use of non-aqueous solvents.

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