Evidence for a three-iron center in a ferredoxin from Desulfovibrio gigas. Mössbauer and EPR studies.

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with Mössbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and Mössbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mössbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796).

center containing 3 iron atoms (and acid-labile sulfur). For this protein, a detailed data analysis was impeded by the presence of an Fe4S4 center. Therefore, the characterization of the new iron-sulfur cluster required a rather unusual data analysis. Here, we report that the new center is also present in a ferredoxin, termed Fd 11, from Desulfovibrio gigas. Since the latter protein contains only one type of metal center, it affords a precise data analysis.
D. gigas ferredoxin is isolated in different oligomeric forms. Fd' I1 is a tetramer of identical polypeptide subunits, each monomer having 57 amino acids of known sequence (3,4). It has been shown (5) that Fd I1 mediates electron transfer between cytochrome c3 and the sullite reductase system, while another oligomeric form, the trimeric Fd I, serves as a carrier in the phosphoroclastic reaction. The presence of iron and acid-labile sulfur and the EPR characteristics of both Fd I and I1 have suggested (6) the presence of Fe4S4 centers. We will demonstrate below that Fd I1 has a novel chromophore containing 3 iron atoms. The work described here complements in many respects the studies on the Azotobacter ferredoxin (1) which we reported with our co-workers of W. H.

MATERIALS AND METHODS
The conditions for growth of D. gigas and the isolation of the tetrameric form of ferredoxin (Fd 11) have been described previously (7). Throughout this manuscript, iron and spin concentrations will be quoted per monomer. Iron was determined by forming a ferrous complex with 2,4,6-tripyridyl-s-triazine using the procedure described by Fischer and Price (8).
EPR spectra were recorded in Dr. J. D. Lipscomb's laboratory on a Varian E-9 spectrometer fitted with an Oxford Instruments continuous flow cryostat. The Mossbauer spectrometer and methods of data analysis have been described previously (9). All isomeric shifts are quoted relative to iron metal at 295 K.

RESULTS
EPR Studies-We have performed iron analyses of three different Fd I1 preparations and found 2.97,3.11, and 2.95 iron atoms/monomer. The sample used for the Mossbauer and EPR studies had 2.97 iron atoms/monomer; the monomer concentration was determined by amino acid analysis of the sample. (The sample was analyzed for eight stable amino acids; their distribution fits the known sequence.) The Mossbauer data gave no evidence for any iron impurities; this allowed us to determine the number of iron atoms per EPRactive center.
As isolated Fd I1 exhibits a fairly isotropic EPR signal around g = 2. Typical spectra are shown in Fig. 2b of Ref.

6.
We have studied the EPR spectra of Fd I1 extensively in the temperature range from 2 to 12 K. These studies suggest strongly that the material is homogeneous, i.e. only one EPRactive species is present. By quantitating the spectra at 6, 8, and 12 K against a copper-EDTA standard, we found 0.93 _+ 0.12 spins/3 iron atoms. The quoted uncertainty is mainly due to the problem of taking EPR spectra of the sample and copper-EDTA at precisely the same temperature.
We have also performed spectral simulations of the EPR spectra. A good representation of the observed spectra was obtained by choosing gl = 2.02, gz = 2.00, and gs = 1.97, using gaussians of widths 15, 35, and 80 G at gl, gz, and g3, respectively. At present, these values are tentative.
Mossbauer Results- Fig. L4 shows a Mossbauer spectrum of oxidized Fd I1 taken at 77 K. The solid line is a least squares fit to the spectrum assuming that each of the three irons yields the same spectrum. The quality of the fit, the symmetry of the spectrum, and the sharpness of the absorption lines (0.28 mm/s full width) support this assumption. The parameters for the quadrupole splitting, A& = 0.54 f 0.03 mm/s, and the isomeric shift, 13 = 0.27 f 0.03 mm/s are practically the same as those observed for rubredoxin (lo), suggesting a tetrahedral coordination of (predominantly) sulf u r atoms.  The subspectra of Sites 1 and 2 are nicely resolved for the Azotobacter protein, while the spectrum of Site 3 is masked by the absorption of the high potential center. In Fd 11, the resolution between Sites 1 and 2 is poorer because the internal magnetic fields at these sites differ less. The spectrum of Site 3, on the other hand, is clearly discernible. Taken together, the data reveal three distinct sites. We have computed theoretical spectra of the three subcomponents (see Fig. 2 In Equation 1, all symbols have their conventional meaning; the g-tensor is known from the EPR data. The spectrum of Site 1 (Fig. 2, solid line) has essentially the same magnetic splitting (A, = 27 MHz, Ay = A , = 44 MHz) as the corresponding one of the Azotobacter ferredoxin. The splitting of the Site 2 spectrum (Fig. 2, dashed line) is substantially larger in Fd I1 (A, = 29 MHz, A, = A , = 16 MHz), accounting for the poorer resolution. As suggested previously (l), the spectrum of Site 3 (Fig. 2, dotted line)  The iron associated with Doublet I1 is high spin ferric in character. For the ferredoxin from A . u i n e h d i i , we have listed arguments that Doublet I1 must be part of a spincoupled structure (1). This contention is further supported by studies of reduced Fd I1 in applied magnetic fields. We have studied the reduced Fd I1 sample at 4.2 K in fields up to 60 kG; a spectrum taken in a 10-kG field is shown in Fig. 3.
The most distinctive property of reduced Fd I1 is that a field of only a few hundred gauss elicits substantial broadening of both doublets due to induced magnetic hyperfine interactions. This unusual behavior suggests strongly that.the magnetic spectra of both Sites I and I1 are controlled by the same electronic spin S. The broadening proves that reduced Fd I1 is paramagnetic, i.e. S > 0. The features of the observed spectra allow us to draw the following conclusions: 1) The electronic spin relaxation rate at 4.2 K is slow compared to the nuclear precession frequencies.
2) The unknown electronic spin S is an integer. 3) Since the magnetic hyperfine fields saturate already in weak applied fields, the lowest electronic spin levels are two closely spaced states of energy separation A (we found A = 0.35 cm").
Analysis of the data reveals that the two equivalent iron sites of Doublet I remain indistinguishable in strong applied fields, i.e. the spectrum in Fig. 3 is a superposition of two spectra with an intensity ratio of 2:l. Furthermore, the iron nucleus associated with Site I1 experiences a positive internal magnetic field, proving this nucleus to be a member of a spincoupled structure. The line assignments of the spectrum in A Three-Iron Center in a Ferredoxin from D. Gigas values for AEQ and S of the latter compound match those found for Site I; the high field spectra have a close resemblance also. The electronic system has an easy axis of magnetization and in applied fields up to 10 kG the Mossbauer spectra of Sites I and I1 are characterized by internal fields of -237 kG and +250 kG, respectively. In stronger fields, anisotropies of the magnetic hyperfine interactions become apparent. The results of spectral simulations are shown in Fig. 3; also shown is a decomposition of the spectrum into subcomponents. Although the fits to the data are excellent, the ambiguity commonly associated with multiparameter fits requires more detailed studies. At present, the main result is the fact that the magnetic behavior of the iron sites is describable by a common electronic spin, i.e. a spin-coupled cluster is strongly suggested.?

DISCUSSION
In the following, we will review the main evidence in support of a spin-coupled cluster containing 3 iron atoms. The present preparations consistently yield 3 iron atoms/monomer (or 12 iron atoms/holoprotein). Our studies show that the material is pure; there is no evidence for iron impurities from either EPR or Mossbauer spectroscopy. The magnetic Mossbauer spectra of oxidized Fd I1 observed at 4.2 K show three distinct iron sites and they attest that the sites belong to EPR-active centers.," The EPR data reveal the presence of only one type of paramagnetic center; a spin quantitation yields 0.9 spins/3 iron atoms. Taken together, the data show that 3 iron atoms belong to the EPR-active center.
Upon reduction by 1 electron, the center becomes EPRsilent ( 7 ) . The Mossbauer data reveal that reduced Fd I1 is paramagnetic, i.e. the electronic ground state of the clusters has S > 0. The Mossbauer spectra demonstrate two distinct iron environments which are present in the ratio of 2:1. The features of the magnetic Mossbauer spectra and their response to applied magnetic fields suggest a common electronic spin, i.e. the sites are subsites of a spin-coupled cluster. Spin COUpling is indicated by the observation of positive and negative magnetic hyperfine fields. Thus, both the oxidized and the reduced states independently yield evidence for a three-iron center.
The observed isomeric shifts suggest that the irons have tetrahedral environments of sulfur atoms. This, however, does not rule out the possibility that a site might have one oxygenic Details about the spectral simulations of the 10-kG spectrum and a set of parameters may be obtained from E. Miinck upon request.
' Strictly speaking, only two sites can be proven directly to belong to EPR-active centers. Since the third site has a small hyperfine interaction the nuclear Am = 0, lines are not resolved. On the other hand, the third site is high spin ferric in character, as attested by the observed isomeric shift. This site must either be a structural component of a spin-coupled cluster or an EPR signal typical of a high spin ferric ion must be observed. Also, if the third site were not part of the cluster, another subsite with half-integral electronic spin has to be postulated; 2 ferric ions cannot spin-couple to form the observed halfintegral system spin.
or nitrogenous ligand no model complexes are available for comparisons. It is interesting to note that the three irons are distinguishable in the oxidized state of the cluster (there are three distinct Mossbauer spectra at 4.2 K). In the reduced state, however, 2 iron atoms (the ones yielding Doublet I) are indistinguishable even in strong applied fields. The increased isomeric shift and the quadruple splitting suggest that the 2 iron atoms of Doublet I share the electron that enters the complex upon reduction; both sites are roughly at the oxidation level Fe+2.5. I t is noteworthy that the Fd I1 spectroscopically resembles closely the Azotobacter ferredoxin; yet, the midpoint potentials of the three-iron centers differ by 280 mV.
Besides the tetrameric Fd 11, the basic subunit can form a trimeric protein, termed Fd I (7). EPR studies of reduced Fd I have elicited signals qualitatively similar to those observed for reduced Fe4S4 centers (6). A Fd I sample appropriate for Mossbauer studies is anticipated with considerable interest.