Azotobacter vinelandii Ferredoxin I

The reduction potential (E 0′) of the [4Fe-4S]2+/+ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is ∼200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences inE 0′ do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S]2+/+clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S]2+/+ cluster with a very negativeE 0′ similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E 0′ clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S]2+/+cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higherE 0′ P. asaccharolyticus ferredoxin all led to increases in E 0′ for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH–S bond between the introduced Ser side chain and the Sγ atom of Cys ligand 42 and an accompanying movement of water.

Both experimental and theoretical research has been directed toward understanding how the polypeptide surrounding the cluster controls the reduction potential. Factors that have been proposed as being important include (a) solvent exposure of the cluster, (b) specific hydrogen bonding networks especially NH-S bonds, (c) the proximity and orientation of protein backbone and side chain dipoles, and/or (d) the proximity of charged residues to the cluster (7,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). This study concerns protein control of the reduction potential of a [4Fe-4S] 2ϩ/ϩ cluster that is ligated via a typical CXXCXXC motif with one remote Cys ligand.
Previous studies in this area have focused on comparing the environments of the clusters in structurally characterized proteins. The [4Fe-4S] 2ϩ/ϩ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) 1 has a low E 0 Ј of about Ϫ620 mV (pH 7.0) (36), whereas the analogous clusters in Peptostreptococcus asaccharolyticus ferredoxin (PaFd) and Clostridium acidiurici ferredoxin have E 0 Ј of about Ϫ430 mV (not pH-dependent) (37). Comparison of the structures of these proteins showed that the peptide folding around the analogous clusters is highly conserved with respect to the location of the four Cys ligands, the Cys dihedral angles, and the eight amide groups hydrogen-bonded to sulfur atoms of the cluster (22). Although early theoretical studies suggested that the solvent access to the [4Fe-4S] 2ϩ/ϩ cluster of PaFd might explain its more positive E 0 Ј value, a more recent 0.95-Å structure of C. acidiurici ferredoxin failed to reveal the presence of internal water molecules (38). The proposal that negatively charged surface residues or hydrophobic residues near the [4Fe-4S] 2ϩ/ϩ cluster of AvFdI might explain its more negative reduction potential (7,(23)(24)(25)(26)(27)(28) was directly tested by converting six individual amino acid residues in AvFdI into the corresponding residue in PaFd (29). Those substitutions, however, failed to raise the E 0 Ј of the [4Fe-4S] 2ϩ/ϩ cluster of AvFdI. Thus, at present both experimental and theoretical approaches have failed to determine what factors are responsible for the 200-mV difference in E 0 Ј for the [4Fe-4S] 2ϩ/ϩ clusters of AvFdI versus PaFd and C. acidiurici ferredoxin.
In this study, we have extended the sequence/structure comparison approach to include a third class of ferredoxins and have achieved substantial increases in the E 0 Ј of the [4Fe-4S] 2ϩ/ϩ cluster of AvFdI by substitution of individual residues.

EXPERIMENTAL PROCEDURES
Mutagenesis of fdxA, Expression, Purification, and Crystallization of FdI Variants-Site-directed mutagenesis was carried out as described elsewhere (29) using the following oligonucleotides: 5Ј-ACCGATTGT-GTTGAAGAGTGCCCGGTAGACTGT-3Ј for V19E; 5Ј-GACTGCGC-GAGCTGCGAGCCCGAGTGC-3Ј for L44S; and 5Ј-GCTCTGCGAGTC-CGAGTGCCCCGCCCAG-3Ј for P47S. Cell growth and the purification and triclinic crystallization of FdI variants were carried out as described elsewhere (39). All mutants accumulated to native levels in the cells.
Protein Characterization-EPR spectra were obtained using a Bruker ESP300E spectrometer, interfaced with an Oxford liquid helium cryostat. For spin quantitation of reduced [4Fe-4S] ϩ , the spectrum of L44S after reduction by 4 mM sodium dithionite was recorded at 15 K using 60 M purified L44S FdI at a microwave power 1 ⁄2 of 1 milliwatt. Under those conditions, the EPR signal of FdI is linearly proportional to (microwave power) 1 ⁄2 . A sample of 50 M Cu 2ϩ -EDTA used as a standard was recorded at the same EPR setting and temperature, where the signal is also linearly increasing in the function of (power) 1 ⁄2 . Absorption spectra were recorded in 0.5-ml quartz cuvettes on a Hewlett Packard 8452A diode array spectrophotometer. CD spectra were obtained using a Jasco J-500C spectropolarimeter. For UV-visible and CD measurements of reduced sample, 4 mM sodium dithionite was added to samples in a Vacuum Atmospheres anaerobic chamber. UV-visible and CD measurements were carried out using 1-ml volume cylindrical cells with fused quartz windows.
Electrochemistry-Purified water of resistivity ϳ18 megaohms-cm (Millipore) was used in all experiments. The buffers MES, HEPES, and TAPS and the co-adsorbate neomycin sulfate were purchased from Sigma. An AutoLab electrochemical analyzer (EcoChemie, Utrecht, The Netherlands) was used to record DC voltammograms. The three-electrode configuration featuring all glass cells has been described previously (40). The sample compartment (typically holding 500 l) was maintained at 0°C to optimize stability. All E 0 Ј values are given with reference to the standard hydrogen electrode. The saturated calomel electrode was held at 22°C, which we have adopted as E (saturated calomel reference electrode) ϭ ϩ243 mV versus standard hydrogen electrode (41). E 0 Ј values from cyclic voltammetry were calculated as the average of the anodic and the cathodic peak potentials, E 0 Ј ϭ 1 ⁄2(E pa ϩ E pc ). The pyrolytic graphite "edge" electrode (surface area typically 0.18 cm 2 ) was polished prior to each experiment with an aqueous alumina slurry (Buehler Micropolish; 1.0 m) and then sonicated extensively to remove traces of Al 2  Structure Determination-Tetragonal crystals of the V19E and P47S FdI mutants were grown as previously described (32). Data for the V19E and P47S mutants were collected using an R-AXIS IV imaging plate, Osmic mirrors, and a Rigaku RU300 rotating anode x-ray source equipped with an Crystal Logic N 2 cryogenic system; data for the L44S mutant were collected at Stanford Synchrotron Radiation Laboratory beam line 7-1. The structures were solved by molecular replacement. The V19E FdI structure was refined to 1.65-Å resolution with R ϭ 0.249 and R free ϭ 0.265; the P47S FdI structure was refined to 1.90-Å resolution with R ϭ 0.240 and R free ϭ 0.266. Monoclinic crystals of L44S FdI were grown as for native FdI with the addition of 200 mM lithium sulfate to the reservoir solution. The space group is C2 with a ϭ 130.8, b ϭ 85.6, c ϭ 67.2 Å, ␤ ϭ 117.9°, and four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to 2.20-Å resolution with R ϭ 0.243 and R free ϭ 0.286. Details of the data collection and refinement for each mutant FdI structure determination are included in the Protein Data Bank depositions. The coordinates for the structures of the V19E, L44S (four independent structures), and P47S mutants of FdI have been deposited in the Protein Data Bank with accession codes 1G3O, 1GAO, and 1G6B, respectively.

RESULTS AND DISCUSSION
Selection of Mutants-Eight-iron ferredoxins containing two [4Fe-4S] 2ϩ/ϩ clusters have long been known to fall into two general classes with respect to sequence motifs. The "clostridial-type" ferredoxins, represented by the structurally characterized PaFd and C. acidiurici ferredoxin, are M r ϳ5,550 proteins where both clusters receive three of their four ligands from a CXXCXXC motif (1-6, 22, 38, 42, 43). The other class is represented by the structurally characterized, M r 9,058 Chromatium vinosum ferredoxin (CvFd), which has one cluster ligated by a CXXCXXC motif and the other ligated by a CXXCX 8 C motif (42,44). Regardless of these differences in sequence motif, both classes of proteins have long been believed to contain two [4Fe-4S] 2ϩ/ϩ clusters of equivalent E 0 Ј (10, 42). This view was challenged in 1998 by our discovery that a ferredoxin from A. vinelandii (AvFdIII) was very similar to CvFd based on molecular weight and sequence motif and yet had very different E 0 Ј values of Ϫ486 and Ϫ644 mV for its two [4Fe-4S] 2ϩ/ϩ clusters (45). A reexamination of CvFd then led to the conclusion that it also had very different E 0 Ј of about Ϫ460 mV and about Ϫ655 mV for its two [4Fe-4S] 2ϩ/ϩ clusters (46,47). These studies further identified the lower E 0 Ј cluster of CvFd as the one with the CXXCXXC motif.
The similarity of E 0 Ј values for the [4Fe-4S] 2ϩ/ϩ cluster of AvFdI (Ϫ620 mV at pH 7.0) and the lower E 0 Ј clusters of AvFdIII and CvFd led us to extend the sequence and structure comparison to include these proteins. Fig. 1 compares the sequences around the [Fe-S] clusters of PaFd, AvFdI, CvFd, and AvFdIII. Although clearly evolutionarily related (48), PaFd and AvFd vary greatly in size (M r 5,556 versus 12,050) and cluster composition (8Fe versus 7Fe), and they share only 48% sequence similarity in their first 50 amino acids, where the clus-  (29). The amino acids in red represent the residues of interest in this study. Cluster numbers refer to the position of the cluster relative to the NH 2 terminus of the protein.
ters are located. As in previous studies (10,22,29), the homologous [4Fe-4S] 2ϩ/ϩ clusters (i.e. the second cluster from the NH 2 terminus) are being compared, with PaFd having an E 0 Ј value ϳ200 mV more positive than the corresponding cluster in AvFdI. AvFdI and CvFd also vary greatly in size (M r 12,050 versus 9,058) and cluster composition (7Fe versus 8Fe) and share only 42% sequence similarity in their first 50 amino acids, where the clusters are located. In this case, however, it is the first cluster from the NH 2 terminus of CvFd that has a very low E 0 Ј value, similar to the second cluster from the NH 2 terminus of AvFdI. Despite these differences, Fig. 1 shows that the sequences surrounding the two low E 0 Ј clusters in AvFdI and CvFd (or AvFdIII) are extremely similar to each other.
In a previous study, we constructed D23N, F25I, H35D, E38S, and E46A variants of AvFdI (Fig. 1) and found no change in E 0 Ј (29). Based on the sequence comparisons shown in Fig. 1 and based on the protein structures, we have now constructed and characterized V19E, L44S, and P47S variants of AvFdI in attempts to mimic their positions in PaFd for which E 0 Ј is higher. The residues corresponding to Val 19 and Pro 47 are completely conserved in the low E 0 Ј classes of ferredoxins. As discussed elsewhere, the residue corresponding to Leu 44 varies, and in the low E 0 Ј ferredoxins, it is generally Leu or Val or occasionally Met (46). The selection of these mutations was further encouraged by theoretical and experimental studies showing positive changes in E 0 Ј induced by substitution of nonpolar residues (e.g. Leu and Val) by polar residues, including serine (32,49). Fig. 2A is an overlay of the homologous [4Fe-4S] 2ϩ/ϩ clusters of CvFd and AvFdI, showing their similarity in this region. Fig. 2B is an overlay of the same region of AvFdI and PaFd, showing the positions in the native proteins of the three residues under consideration relative to the cluster.
Purification and Characterization of the FdI Variants-The V19E, L44S, and P47S variants of AvFdI were constructed, expressed in their native background in A. vinelandii, and purified as described under "Experimental Procedures." These variants were all air-stable and accumulated to levels similar to that of native FdI in the cell. Fig. 3 compares the UV-visible absorption spectra of each of the variants with those of native FdI, both in their air-oxidized states and following the anaerobic addition of dithionite. The oxidation states of the clusters in the presence of air are [4Fe-4S] 2ϩ and [3Fe-4S] ϩ . For native FdI, dithionite reduces only the [3Fe-4S] ϩ cluster to the "zero" oxidation level but does not reduce the [4Fe-4S] 2ϩ cluster because its reduction potential is too low (36,50). This result is also obtained for V19E and P47S variants. In contrast, as shown in Fig. 3, dithionite causes considerably more reversible bleaching of L44S, suggesting that the E 0 Ј value for its [4Fe-4S] 2ϩ/ϩ cluster has increased relative to native FdI. This reduction of the [4Fe-4S] 2ϩ cluster in L44S was confirmed by EPR spectroscopy.
As shown in Fig. 4, air-oxidized FdI exhibits a characteristic g ϭ 2.01 EPR signal that arises from the [3Fe-4S] ϩ cluster having S ϭ 1 ⁄2. Val 19 , Leu 44 , and Pro 47 are all remote from that cluster, and, as expected, the mutation of these residues did not influence that signal. Consistent with the data shown in Fig. 3, the anaerobic addition of dithionite to V19E and P47S FdIs led to reduction of the [3Fe-4S] ϩ cluster to the "zero" oxidation level and disappearance of the g ϭ 2.01 signal but did not result in reduction of the [4Fe-4S] 2ϩ cluster, the same result that is obtained for native FdI (36). For L44S, the [4Fe-4S] 2ϩ cluster could be partially reduced by dithionite (Fig. 4), thereby confirming the conclusion from the UV-visible absorption experiment (Fig. 3). Spin quantitation of the signal of the [4Fe-4S] ϩ cluster using Cu-EDTA showed that 20% of the L44S [4Fe-4S] 2ϩ cluster was reduced by dithionite, consistent with the E 0 Ј data discussed below. The wavelength dependence and form of the CD spectra of oxidized and reduced native FdI and V19E, L44S, and P47S variants are extremely similar (Fig. 5). In previous studies of variants that showed structural rearrangements in the vicinity of the [4Fe-4S] 2ϩ/ϩ , the CD spectra were dramatically different (36,49,51). Thus, the CD data, combined with the UV-visible and EPR strongly suggest that the overall protein folding in solution and the environment around the clusters has not been significantly perturbed in any of the altered proteins.
Reduction Potential Measurements-The E 0 Ј values for the [Fe-S] clusters of native FdI, V19E, L44S, and P47S were measured under identical conditions using voltammetric methods as described under "Experimental Procedures." Results obtained at pH 7.0, for ferredoxin solutions containing 4 mM neomycin, are shown in Fig. 6. Two pairs of well defined oxidation and reduction peaks are observed in each case, with the higher E 0 Ј peaks corresponding to oxidation/reduction of the [3Fe-4S] ϩ/0 cluster and the lower E 0 Ј peaks corresponding to oxidation/reduction of the [4Fe-4S] 2ϩ/ϩ cluster (40). The mutations were all remote from the [3Fe-4S] ϩ/0 cluster, and, as expected from previous studies (e.g. see Refs. 29 and 36), no changes were observed in E 0 Ј for that couple. The [4Fe-4S] 2ϩ/ϩ cluster E 0 Ј of native FdI is known to be weakly pH-dependent, and the same pH dependence was observed for all of the variants examined in this study. Table I summarizes the data collected at pH 7.0 and shows that in all three cases positive changes in E 0 Ј were observed. For V19E, the increase was 13 mV; for L44S, the increase was 94 mV; and for P47S, the increase was 23 mV. The electrochemical measurements for these mutants have an error of about Ϯ10 mV.
X-ray Structures of the Variants-X-ray structures of all three variants were obtained in order to determine whether the intro-duced residues in AvFdI adopted the same orientations as in native PaFd and thus help interpret the observed changes in E 0 Ј. V19E- Fig. 7A compares the structure of V19E, refined to 1.65 Å, with that of native FdI in the region of the [4Fe-4S] 2ϩ/ϩ cluster, and Fig. 7B compares the orientation of Glu 19 in V19E with that of the corresponding residue in PaFd. Overall, the structure of V19E is very similar to that of native FdI, and there is virtually no difference between the native FdI and V19E structures at the position of Cys ligand 20. There is also no change in solvent accessibility as evidenced by no change in the packing around the [4Fe-4S] 2ϩ/ϩ cluster. When compared with PaFd, the introduced Glu is not exactly in the same orientation (Fig. 7B), but in both cases the distances to the cluster are very long, with the shortest distance from the Glu 19 O⑀1 to the nearest sulfur atom of the [4Fe-4S] 2ϩ/ϩ cluster being 7.8 Å for V19E versus 8.6 Å for PaFd. Consistent with the similarities of the native FdI and V19E structures, V19E gave a barely perceptible positive increase in E 0 Ј relative to native FdI. The fact that introducing a negatively charged surface residue, with a carboxylate to cluster distance of 7.8 Å, does not result in a significant change in E 0 Ј is fully consistent with the initial study on surface charged residues (29).
P47S- Fig. 8A compares the structure of P47S, refined to 1.9 Å, with that of native FdI in the region of the [4Fe-4S] 2ϩ/ϩ cluster, whereas the structure is indistinguishable from that of native FdI. In this case, a small 23-mV increase in E 0 Ј was obtained, possibly due to the introduction of an OH dipole that is directed toward the cluster, albeit at a long distance. Three new ordered water molecules are also observed in P47S versus native FdI between the Ser 47 side chain and the carbonyls of Ala 43 and Leu 44 , but these do not increase solvent accessibility, since the packing around the cluster remains the same. (Note that these are also much farther from the [4Fe-4S] 2ϩ/1ϩ cluster, at 8 -10 Å, versus the new waters in L44S, which had a much greater increase in E 0 Ј (see below).) As for V19E, the P47S mutation occurs in a single turn of helix linking the two clusters. The main chain conformation is somewhat different in native FdI versus PaFd (Fig. 8B), with a difference in the C␣ positions at the site of mutation of ϳ1.6 Å. Consequently, the C␣-C␤ orientation of Ser 47 in P47S FdI is different from that of Ser 44 in PaFd, but due to the conformation of the Ser 47 side chain, the O␥ atoms of the two Ser are fairly close in space, 1.43 Å apart in the superposition. The closest distance to the [4Fe-4S] 2ϩ/ϩ clusters also remains about the same at 9.3-10 Å. Thus, like V19E, the P47S mutation makes AvFdI more like PaFd without significant structural perturbation. Because each mutant mimics PaFd better, this is consistent with the slightly higher E 0 Ј in both cases.
Ϫ596 ϩ23 mV, consistent with the proposal that this position is a critical determinant for E 0 Ј (46). Its structure was refined to 2.2-Å resolution and revealed that there were four copies in the asymmetric unit in two "dimers" in the C2 space group. The density for the four copies at Ser 44 varies from very strong to weak, but all four are modeled with confidence. Fig. 9A compares the four copies with each other, and Fig. 9B compares copy A to native FdI in the vicinity of the [4Fe-4S] 2ϩ/ϩ cluster. The four independent copies of L44S are very similar, with root mean square deviations of 0.24 -0.39 Å overall. However, there is some variation at the site of the mutation, apparently accounting for the four copies in the crystallographic asymmetric unit. This suggests that the Leu-to-Ser substitution in the context of the AvFdI sequence (Cys-Ala-Leu/Ser-Cys) versus the PaFd (Cys-Gly-Ser-Cys) sequence is not energetically favorable, giving rise to the alternate conformations. formed. For copies A, B, and D, the geometry and contacts are consistent with a hydrogen bond of 3.4 -3.6 Å from the serine hydroxyl to S␥ of Cys 42 . The conformation of these side chains is very similar to the corresponding Ser in PaFd as illustrated in Fig. 9C, which compares the structure of that protein to copy A of L44S Fd. As shown, the same hydrogen bond occurs in PaFd (new Protein Data Bank file 1DUR). Although the contact between the O␥ of Ser 44 and the S␥ of Cys 42 is roughly orthogonal to the C␤-O␥ and C␤-S␥ bonds, the hydrogen atom on O␥ can point almost directly at the S␥, because unlike a main chain amide, it is bonded to the O␥ at 109°(sp 3 bond). The introduction of this dipole in at least three-fourths of the copies (if the crystal conformations are averaged in solution) is clearly the primary factor in the elevated E 0 Ј of PaFd versus native AvFdI or L44S versus native FdI.
In addition to the introduction of an OH-S bond, the presence of the Ser side chain results in ordered water molecules involved in hydrogen bonds with Ser 44 and adjacent residues (3, 1, 0, and 2 additional water molecules in copies A, B, C, and D, respectively, compared with native FdI). While not in direct contact with the atoms of the [4Fe-4S] 2ϩ/ϩ cluster, the dipole moments of these ordered water molecules near the cluster could also serve to elevate E 0 Ј. In particular, in copies A, B, and D, a water molecule bridges between Ser 44 O␥ and the carbonyl of Val 19 and is 4.2-5.6 Å from the S␥ of Cys 42 , the closest atom. Therefore, the presence of a Ser at position 44 not only provides a direct hydrogen bond dipole to the cluster but also provides secondary dipoles due to additional ordered water molecules at the protein surface. A water molecule at the homologous position, bridging Ser 42 to the carbonyl of Glu 17 , is also observed in PaFd, in this case 5.8 Å from S␥ of Cys 39 , whereas no water is observed proximal to Leu 44 in native AvFdI, consistent with the hypothesis.
It should be noted that all of the proteins compared here have an NH-S␥ hydrogen bond from residue 44 (AvFdI)/41 (PaFd)/13 (CvFd) to Cys 42 /Cys 39 /Cys 11 ; the average distance in the four copies of L44S (3.45 Å) is comparable with that in native FdI and PaFd (3.5 Å) and similar to CvFd (3.8 Å), so this and other features of the hydrogen bonding around the [4Fe-4S] 2ϩ/ϩ cluster are not perturbed in the mutant and cannot be responsible for the increase in E 0 Ј.
A recent study of CvFd suggested that the presence of a bulky residue at a position corresponding to position 44 of native FdI is primarily responsible for the very low reduction potential of the [4Fe-4S] 2ϩ/ϩ clusters found in CvFd, AvFdIII, and AvFdI (46). The data presented here suggest instead that in the case of PaFd an ϳ100 mV increase in [4Fe-4S] 2ϩ/ϩ E 0 Ј relative to the corresponding cluster in AvFdI is a direct result of the presence of the OH-S bond from the serine hydroxyl to the Cys 42 ligand, with the positive (hydrogen) end of the introduced CH 2 OH side chain closest to the negatively charged S␥ of the Cys 42 ligand, leading to an increase in E 0 Ј. A secondary effect may also involve the presence of newly bound water molecules at longer distances.
Conclusions-The E 0 Ј of the [4Fe-4S] 2ϩ/ϩ cluster of AvFdI is ϳ200 mV more negative than that of the homologous cluster in PaFd. Previous mutagenesis experiments tested the contribution that individual amino acid residues make to the control of E 0 Ј by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitution of negatively charged surface residues with neutral residues, and two involved substitution of hydrophobic residues, but none resulted in any increase in the [4Fe-4S] 2ϩ/ϩ cluster E 0 Ј. Recently, another class of ferredoxins, represented by AvFdIII and CvFd, was shown to contain [4Fe-4S] 2ϩ/ϩ clusters with E 0 Ј values similar to that of AvFdI (45,46). The observation that the  (Fig. 1) and structures (Fig. 2A) surrounding the very low E 0 Ј [4Fe-4S] 2ϩ/ϩ clusters in the dissimilar AvFdI and AvFdIII/CvFd are extremely similar encouraged the construction of three additional mutations. In this case, all three mutations led to positive increases in E 0 Ј. Although Val at a position corresponding to FdI residue 19 is conserved in the low E 0 Ј classes of ferredoxins, and Glu is highly conserved at the corresponding position in the higher E 0 Ј ferredoxins, the minor change in E 0 Ј for V19E shows that this residue is not a critical determinant of E 0 Ј. A still small, but somewhat larger, change in E 0 Ј of ϩ23 mV was obtained for P47S by mimicking PaFd without modification of the FdI structure except at that position, possibly due to the introduction of the serine dipole and associated water molecules at long distances from the cluster. The largest change was observed for L44S for which an increase of 94 mV was obtained; here the environment of PaFd was reproduced by introducing an OH-S hydrogen bond from the serine hydroxyl to the cluster ligand Cys 42 with accompanying movement of water. Since the residue at position 44 is not conserved in either the high or the low E 0 Ј classes of [4Fe-4S] 2ϩ/ϩ clusters, future experiments will be aimed at determining the influence that other residues at this position have on reduction potential.