High and low reduction potential 4Fe-4S clusters in Azotobacter vinelandii (4Fe-4S) 2ferredoxin I. Influence of the polypeptide on the reduction potentials.

Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv. It exhibits and EPR signal of g equals 2.01 in its isolated form. This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin. A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP). On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed. This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum. These characteristics suggest that a cluster in A. vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic. The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different.

suggest that a cluster in A. oinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichiometry studies showed that only one 4Fe-4S* cluster in this (4Fe-4S*), ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I that shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic.
The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. uinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (-+340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster i n Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are -760 mv different.
Azotobacter uinelandii, a nitrogen-fixing aerobic bacterium, has been shown to contain a number of iron-sulfur proteins (1). In addition to the nitrogenase, which is an iron-sulfur protein complex, this organism contains two 2Fe-2S* proteins (called Azotobacter iron-sulfur proteins I and II (2)) and two proteins containing one or more 4Fe-4S* clusters (3-5). An 8-Fe protein (two 4Fe-4S* clusters) previously referred to as either ferredoxin I (Fd I) (5) or iron-sulfur protein III (4), has been obtained in crystalline form with M, = 14,500 and a midpoint reduction potential (E,,) of -420 mv (5). This A. uinelandii protein will be referred to as A. uinelandii (4Fe-4S*), Fd or Fd duction of NADP by spinach chloroplasts and it will couple the reducing power of illuminated spinach chloroplasts to Azotobatter nitrogenase.
In addition, Fd I functions with low activity in the clostridial phosphoroclastic reaction (5). Its optical spectrum resembles that of other ferredoxins with cIoO = 27,000 M-' cm-' and an A,,,:AzaO value of 0.58 (5).
The protein can replace spinach ferredoxin in the photore-'Results described in this paper indicate that separation of iron-sulfur moteins according to their reduction potentials may not he *This work was supported by Research Grant A-2109 from the advisable. Although the term High-Potential-Iron-Protein (HiPIP) National Institute of Arthritis and Metabolic Diseases, United States does convey useful information, we feel that the name Chromatium oxidized P. aerogenes ferredoxin (6) and both oxidized and acidi-urici ferredoxin were used as spin standards. The intensity of the reduced Chromatium HiPIP (11,12) have shown that, the EPR signal at g = 2.01 exhibited by A. vine&&i Fd I, observed from iron-sulfur clusters in both proteins are cube-like structures 9.8-19.4 K, was found to obey the Curie Law as is required in order to bonded to the polypeptide chain through a cysteinyl sulfur obtain a meaningful number of unpaired electron spins.
bond to each iron atom. These clusters will be denoted as RESULTS

AND DISCUSSION
FerSr*SrCYs]. In view of their very different reduction potentials, the similarity in the structure of the iron-sulfur clusters Reducible Cluster-The EPR spectrum of Azotobacter uinein the two proteins is surprising. It has been proposed that the landii Fd I (Fig. 1) is almost identical with that exhibited by cluster may exist in three different oxidation states, with oxidized Clostridium Fd (18,23,24 this schen;e, [Fe,S,*S,cYs]-I is the cluster is oxidized HiPIP, The oxidation states of the clusters in super-oxidized and -2 is the charge on the clusters in reduced HiPIP and oxidized oxidized Clostridium ferredoxin have been shown to be the ferredoxin, and -3 is the charge on the clusters in reduced same as those in oxidized and reduced Chromutium HiPIP ferredoxin. The use of net oxidation numbers reflects a lack of (18). The implication is that the cluster in Fd I giving rise to this EPR behavior functions between the same pair of oxidaknowledge of the details of the electronic structure of the tion states as HiPIP cluster. The EPR behavior shown by the 8Fe-8S* ferredoxin (Fd I) from A. uinelundii appears to differ from other 8Fe-8S* Results of quantitative EPR experiments were used to ferredoxins. Shethna (4) has reported an EPR spectrum of the calculate the reduction potential of Fd I, and these results were dithionite-treated Fd I that is similar to the g = 2.01 resonance I I arising from super-oxidized Clostridium Fd (18). Experiments in this laboratory indicated that the g = 2.01 resonance is exhibited by A. oinelundii Fd I in its isolated form. In fact, this resonance is found to be of much greater intensity in the isolated protein than is the resonance exhibited by the F As has been published previously (5), E,, = -420 mv when the ratio [(4Fe-4S*)red]: [(4Fe-4S*)ox] was determined optically. If this ratio is calculated on the basis of quantitative EPR, a reduction potential of -450 mv is obtained. This shows that these EPR states are truly coupled to the reduction of the protein rather than being artifacts resulting from treatment with a reductant. The 30-mv difference between the optically and EPR-determined potentials may result from a temperature dependence of the potential. Optical spectra were recorded at room temperature while EPR spectra were observed at 10 to 20 K. A similar difference was found between the EPRand optically determined reduction potentials of spinach ferredoxin (27).
The EPR results (Fig. 1) show the occurrence of an Fe-S* cluster in A. uinelandii Fd I in its isolated state that is analogous to the clusters in ferricyanide-treated Clostridium ferredoxin. This Fd I cluster has a formal valence of minus one that is identical with that of the cluster in oxidized HiPIP. Therefore, on reduction, this Fe-S* cluster in Fd I functions between the same pair of oxidation states as does the cluster of HiPIP. However, the reduction potential of A. uinelandii Fd I is -420 mv, as opposed to +350 for Chromatium HiPIP. This 770-mv difference constitutes direct evidence of the involvement of the polypeptide in determining the reduction potential of this oxidized-reduced ~e,S,*S,CYS]-1/~e,S,*S,C~s]-2 couple.
Because Fd I is an 8Fe-8S* protein, it is assumed to have two 4Fe-4S* clusters, indicated by the designation [4Fe-4S*],Fd. This view is substantiated by the similarity in the position of the maxima in the optical spectra of A. vinelandii Fd I, Clostridium acidi-urici Fd, and Bacillus polymyxa Fd I, and by their similar molar extinction coefficients per cluster near 400 nm (approximately 4000 Mm 1 cm-' per iron atom) (5, 28,28). In addition, these three proteins exhibit an EPR resonance at g = 2.01, a signal which is not exhibited by any other iron-sulfur protein known to have a different cluster structure.
A. uinelandii (4Fee4S*), Fd I may be expected to accept 2 electrons on reduction because it has two 4Fe-4S* clusters. The isolated form of the protein exhibits an EPR spectrum characteristic of FerS,*S,cYs 1-l (oxidized) clusters. Because the reduction potential is so negative (E,, = -420 mv), both clusters would be expected to be oxidized ( Fe,S,*SrCYs]-l) in the isolated protein. Since the minus one state exhibits the g = 2.01 signal, the EPR spectrum of the isolated form of Fd I should display an intensity equivalent to 2 unpaired (spin = Yz) electrons. However, quantitative EPR experiments show an intensity equivalent to only 1.0 unpaired electron spin ( Table  I), suggesting that only 1 electron is taken up on reduction (i.e. only one cluster is functioning on reduction). To verify this result, the ferredoxin was photoreduced with heated spinach chloroplasts using ascorbate-reduced 2,6-dichlorophenol indophenol as the source of electrons. The light was then turned off and NADP was added anaerobically resulting in the reoxidation of the ferredoxin and a concomitant reduction of the NADP. By determining optically at 340 and 420 nm the molar ratio of NADP reduced to ferredoxin reoxidized, the number of electrons transferred per mol of ferredoxin could be calculated (21). The results are shown in Table I. The average value of 0.92 electron transferred per Azotobacter Fd I molecule obtained in this manner is in good agreement with the value of 1.0 spin obtained by quantitative EPR (Table I). Results obtained with spinach and clostridial ferredoxins are included in Table I as controls. (Two electrons per mol were expected for clostridial ferredoxin, and the observed number of electrons transferred is within 16% of the expected number. One electron per mol was expected to be transferred by spinach ferredoxin and 1.03 electrons per mol were found.) These data indicate that only one cluster in Fd I is functioning on reduction with hydrogen/ hydrogenase, dithionite, or on photoreduction with spinach chloroplasts.

Oxidizable
Cluster-Addition of potassium ferricyanide (K,Fe(CN),) to A. uinelandii (4Fe-4S*), ferredoxin I causes a change in both the optical spectrum (most apparent above 475 nm, see Fig. 3) and the EPR spectrum (Fig. 4). While the The number of electrons transferred on reoxidation was determined by experiments in which the ferredoxins were photoreduced by spinach chloroplast fragments and reoxidized by addition of excess NADP to the reaction mixture. By determining optically at 340 and 420 nm the molar ratio of NADP-reduced to ferredoxin-reoxidized, the number of electrons transferred per mol of Fd I could be calculated. after addition of dithionite to 6.6 x lo-' M, followed by air reoxidation. 7845 change in the optical spectrum is not large, it is reproducible and reversible. The calculated optical difference spectrum exhibits a maximum between 450 and 425 nm. The low and high field shoulders of the g = 2.01 resonance evident in the EPR spectrum of ferricyanide-treated Fd I may result from spin-spin interaction.
No half-field transition indicative of spin-spin interaction was observed in the EPR spectrum, but this may be a result of the expected low transition probability for this resonance. Quantitative EPR of the spectrum of the ferricyanide treated Fd I showed an intensity equivalent to 2.1 unpaired electron spins in the doubly integrated EPR spectrum. This implies that the second cluster in Fd I has given up an electron on oxidation and becomes paramagnetic. This oxidation is reversible, as shown by recovery of 80 to 95% of the optical absorbance at 400 nm (Fig. 3) and a return to the initial EPR spectrum of the protein. To demonstrate the reversibility of the ferricyanide-treated Fd I, it was necessary to reduce the protein with dithionite and then air-reoxidize it to obtain the isolated form of the protein.
Because of the reaction of the Fd with the ferricyanide is slow, and there was some loss of protein upon treatment with ferricyanide, it was only possible to obtain an estimate of the reduction potential.
The potential, estimated optically, was found to be +340 f 50 mv. Care was taken to account for the variable midpoint potential of the ferricyanide/ferrocyanide couple (30). No oxidation of Fd I by p-benzoquinone was observed.
On the basis of these results we believe that the following processes are occurring in A. vinelandii (4Fe-4S*) 2 ferredoxin. 1. On reduction one of the two 4Fe-4S* clusters in the protein accepts an electron: Fe,S,*S,CyS]-l + e-F? ~e,S,*S,CYS]-2 and that this reduction involves the same pair of oxidation states (-1 and -2) as in the reduction of HiPIP. This conclusion is supported by a comparison of the optical spectra of these iron-sulfur proteins showing (Fig. 2) that the extent of change on reduction of A. uinelandii Fd Ferricyanide treated: after treatment with 6-fold excess of K,Fe(CN),. exhibited by Chromatium HiPIP (~ 1 to -2) rather than that of B. polymyxa Fd I (-2 to -3). It is important to remember that two clusters are contributing to the observed spectrum of Fd I while only one cluster is being reduced.
2. On treatment with K,Fe(CN), the second cluster is oxidized while the reducible cluster remains unaffected. This is established by lineshape change in the EPR spectrum observed on oxidation (Fig. 4) and that an intensity equivalent to two unpaired electron spins is found in the oxidized spectrum by quantitative EPR experiments. The oxidation states between which the second iron-sulfur cluster functions are not known with certainty, but the probable states can be inferred as follows. Because the oxidizable cluster exhibits no EPR signal in the isolated form of the protein and exhibits an EPR signal on oxidation, it must function between either the (-4 and -3) or (-2 and -1) oxidation states (the -4 and -2 states are not expected to exhibit EPR spectra while EPR signals are expected from the -3 and -1 states). Only those oxidation states which are stable in the model compounds have been considered. It is not possible to select the correct pair of states by looking at the EPR spectrum of ferricyanide-treated Fd I because this spectrum is not characteristic of any known oxidation state.2 However, because there is no evidence for the -4 state in any iron-sulfur protein, the -2 and -1 oxidation states are assigned tentatively to the ferricyanide-oxidizable cluster in A. uinelandii Fd I. The assignment of this pair of oxidation states (Table II)  iron-sulfur cluster in Chromatium HiPIP and both the high and low potential iron-sulfur clusters in A. uinelandii (4Fe-4S*), ferredoxin I function between the -1 and -2 oxidation states. However, the potentials of these clusters are +350 mv (9, IO), -+340 mv, and -420 mv (5), respectively. Clearly there is significant polypeptide influence on the reduction potential of the ~e,S,*S,CYS]~1/~e,S,*S,C~s]-2 couple in these proteins.
There are at least three possible mechanisms by which the polypeptide could exert this influence. One mechanism would require that the cluster have slightly different geometries in its two oxidation states. This has, in fact, been demonstrated for the cluster in Chromutium HiPIP by x-ray studies (12). The magnitude and sign of the reduction potential depends on the difference in energy of the two oxidation-reduction states. One might envision the polypeptide restricting the geometry of the cluster in one of the oxidation states to a high energy form, and thereby modifying the reduction potential.
A second possible mechanism of polypeptide influence would *This is not surprising, because, for example, [Fe,S,*S,Cy"]ml exhibits different EPR spectra in oxidized HiPIP and super-oxidized Clostridium ferredoxin.
be formation of an additional bond between the cluster and the polypeptide in one of the oxidation states. Thus, a bond formed in the -1 oxidation state would lower the energy of this state, leading to a more negative reduction potential.
Finally, it is possible that the two clusters are close enough together to allow strong electronic interaction.
The presence of spin-spin interaction suggested by the EPR spectrum of K,Fe(CN),-treated Fd I would be consistent with a small intercluster separation. However, spin-spin interaction has been shown to be present between the two clusters in fully reduced clostridial type ferredoxins (31), and both clusters in these proteins are low potential.
Our data do not allow us to choose among these possible mechanisms. CONCLUSIONS A. uinelandii (4Fe-4S*) 2 ferredoxin I represents a previously unrecognized type of iron-sulfur protein. The properties of this protein as described here suggest that: (a) classification of iron-sulfur proteins by their reduction potentials may not be sufficiently versatile. The oxidation-reduction properties of Fd I suggest that the iron-sulfur cluster structure is a more fundamental and useful property for classification of these proteins. Thus, 4Fe-4S* high-potential-iron-proteins and 4Fe-4S* ferredoxins would fall in the same class of protein, rather than being in separate classes, as is currently accepted. (b) The polypeptide can influence the reduction potential of iron-sulfur proteins not only by selecting the functioning oxidation-reduction couple (( ~ 1 + -2) or (-2 + -3)), but also by exerting a direct influence on the potential of the selected couple. Because the oxidized-reduced [Fe,S,*S,C~s]-1/~e,S,*S,c~s]~2 couple exhibits a reduction potential of +350 mv in Chromatium HiPIP and a -420 mv potential in A. uinelandii Fd I, the polypeptide chain can influence the reduction potential of the (-1 + -2) couple by at least 770 mv, and thus determine the potentials at which these electron transfer proteins function.