Y13C Azotobacter vinelandii Ferredoxin I
A DESIGNED [Fe-S] LIGAND MOTIF CONTAINS A CYSTEINE PERSULFIDE*

(Received for publication, January 10, 1997, and in revised form, March 3, 1997)

Mary A. Kemper Dagger , C. David Stout §, Sarah E. J. Lloyd §, G. Sridhar Prasad §, Sarah Fawcett , Fraser A. Armstrong , Binghui Shen Dagger par and Barbara K. Burgess Dagger **

From the Dagger  Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, § Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, and  Department of Chemistry, Oxford University, Oxford, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.


INTRODUCTION

Azotobacter vinelandii ferredoxin I (AvFdI)1 is a Mr 12,000 monomer that contains one [3Fe-4S]+/0 cluster and one [4Fe-4S]2+/+ cluster. The sequence for the NH2-terminal half of AvFdI is homologous to the sequences of the much smaller clostridial-type ferredoxins that contain two [4Fe-4S]2+/+ clusters (1). Comparisons of these and many other [Fe-S] protein sequences have shown that the presence of a highly conserved CysXXCysXXCys motif can be used to predict that a [4Fe-4S]2+/+ cluster will be present (for reviews, see Refs. 2-6). During the evolution of the 7Fe ferredoxins from the 8Fe ferredoxins, two residues were inserted into this motif between the second and third cysteines to form a CysXXCysXXXXCys motif (1). This insertion moved the second cysteine out of reach of the cluster resulting in the inability to form a [4Fe-4S]2+/+ cluster and the appearance of a [3Fe-4S]+/0 cluster in that position (7-13).

The structural consequences of inserting the two additional residues between the second and third cysteines are shown in Fig. 1, which compares the structure of native AvFdI (7-9) to that of Peptococcus aerogenes ferredoxin (PaFd) (10-13) in the [3Fe-4S]+/0 cluster region of AvFdI, which has the sequence Cys8XXCys11XXXXCys16. The comparison in Fig. 1 reveals that the chain trace of PaFd is puckered out in AvFdI, leaving the AvFdI Cys11 removed away from the position of the ligating cysteine in PaFd. Cys11 could therefore not be used as a cluster ligand without substantial structural rearrangement. A further structural difference in this region is that the COOH-terminal half of AvFdI, which is absent in the much smaller PaFd, wraps around the AvFdI residue 8-16 loop shielding it from solvent (9). In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ cluster in the [3Fe-4S]+/0 location of native AvFdI by providing the correct three dimensional orientation of cysteine ligands. As shown in Fig. 1, Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in PaFd so here we report the purification and characterization of Y13C FdI.


Fig. 1. The structure of a CysXXCysXXCys loop in P. aerogenes 8Fe ferredoxin (PaFd), the structure of the corresponding CysXXCysXXXXCys loop in A. vinelandii 7Fe ferredoxin (AvFdI), and a model for the Y13C mutant of FdI (see text). Only side chains of Cys residues and Tyr13 of AvFdI are shown in addition to the [Fe-S] clusters. Carbon, nitrogen, oxygen, sulfur, and iron atoms are shaded white, light gray, gray, dark gray, and black, respectively.
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EXPERIMENTAL PROCEDURES

Materials

Native AvFdI was purified as described previously (14). Ammonium sulfate was from Fisher and all other materials were obtained from the vendors listed previously (15).

Mutagenesis of fdxA and Expression and Purification of Y13C FdI

The oligonucleotide used for the mutagenesis had the sequence GCAAGTACTGCCATTGTGTTGGTGCAAGTGCACCGATTG. This sequence differs from the wild-type sequence (16) by the change of codon 14 from TAC to TGC, resulting in the change of FdI residue 13 from a tyrosine to a cysteine. The success of the mutagenesis was confirmed at the DNA level by dideoxy-DNA sequencing and at the protein level by NH2-terminal protein sequencing (which was carried out in the protein sequencing facility in the department of Molecular Biology and Biochemistry at the University of California, Irvine) and by x-ray crystallography. Unless otherwise indicated, oligonucleotide-directed in vitro mutagenesis (15), FdI overexpression (17), and cell growth (15) were performed as described previously. The purification of Y13C was carried out anaerobically using a modification of purification method two described by Stephens et al. (14). A. vinelandii containing the overexpression vector for Y13C FdI was grown and cell-free extracts were prepared from 1.1 kg of cell paste. No sodium dithionite was used in the purification of Y13C FdI because it became obvious after repeated attempts to purify the protein according to the native FdI purification scheme that the presence of 2 mM sodium dithionite in the buffers denatured this variant of FdI. All steps, however, were carried out anaerobically using thoroughly degassed buffers. The cell-free extracts were not heat treated as described in the purification of nitrogenase (18) due to the possibility that Y13C FdI would be heat-labile. Cell-free extracts were loaded onto a 5 cm × 20-cm DE52 cellulose column equilibrated with extensively degassed 0.025 M Tris-HCl, pH 7.4. Following loading and washing, the column was chromatographed with a 0.1 to 0.4 M NaCl gradient (1.6 liters of each, 570 ml/h) in the same buffer. After this initial gradient Y13C FdI was eluted from the column batchwise with 0.5 M NaCl. The ferredoxin fraction was immediately diluted 1:2 in the same buffer without salt and loaded onto a 2.5 cm × 10-cm DE52 cellulose column. After loading, the column was washed with 4 liters of 0.025 M Tris-KCl, pH 7.4, 0.12 M KCl, at a rate of 300 ml/h. A gradient from 0.12 to 0.3 M KCl (800 ml of each) followed, and Y13C FdI eluted close to 0.25 M KCl. The protein was concentrated on a DE52 cellulose column, and 10-ml samples of concentrated protein were then loaded onto a 1 m × 2.5-cm Sephadex G75 superfine gel filtration column equilibrated in 0.025 M Tris-HCl, pH 7.4, 0.1 M in NaCl. Following elution from that column, 40 ml of 70-80% saturated ammonium sulfate in 0.1 M Tris-HCl, pH 7.4, was added slowly with stirring to 13 ml of Y13C FdI. The protein precipitated over the course of 2 h and was centrifuged in airtight centrifuge tubes in a SS34 rotor at 6,000 rpm for 35 min. The A280/A400 ratio of Y13C FdI following ammonium sulfate precipitation was 1.7 and the A280/A260 ratio was 1.2, values very similar to those reported for the native protein (14). The final yield of Y13C FdI was 19 mg.

Spectroscopy

For spectroscopic studies, all samples were prepared anaerobically under argon in a vacuum atmospheres glove box (O2 < 1 ppm) using degassed buffers. Where indicated samples were first concentrated using a Centricon-10 microconcentrator and were then buffer exchanged into 0.1 M potassium phosphate buffer, pH 7.4, or into a mixture of 50 mM PIPES and 50 mM TAPS buffers at pH 6.0 or 8.3. Electron paramagnetic resonance spectra were obtained using a Bruker 300 Ez spectrophotometer, equipped with an Oxford Instrument ESR-9002 liquid helium continuous flow cryostat. Circular dichroism spectra were obtained using a JASCO J720 spectropolarimeter, and UV-visible spectra were obtained with a HP 8452A diode array UV-visible spectrophotometer.

Electrochemistry

Purified water of resistivity ~18 ohm/m (Millipore) was used in all experiments, and each of the reagents was at least analytical grade. DC (staircase) cyclic voltammetry was carried out using an Autolab electrochemical analyzer (Eco-Chemie, The Netherlands). The all-glass cell, electrodes, and application of protein film voltammetry have been described previously (15, 19). The saturated calomel reference electrode was held at 22 °C, and potential values were converted to the standard hydrogen electrode scale by using E (saturated calomel reference electrode) = +243 mV. The pyrolytic graphite edge working electrode (area approximately 0.18 cm2) was prepared for protein film formation by polishing with an aqueous alumina slurry (Buehler Micropolish 1.0 µm) and sonicating extensively in water to remove traces of Al2O3. All voltammetric experiments were carried out in an anaerobic glove box (Belle technology) with the O2 < 2 ppm. To prepare films, ~1 µl of protein solution (typically 100 µM in 20 mM Hepes, pH 7.0, containing 0.1 M NaC1 with 200 µg/ml polymyxin as co-adsorbate) was applied to the surface of the chilled pyrolytic graphite edge electrode using a glass capillary tip. The buffer-electrolyte solution in the cell contained 0.1 M NaCl, 20 mM mixed buffer (5 mM of acetate, Mes, Hepes, and TAPS) and 200 µg/ml polymyxin. Reduction potentials were determined from the average of peak positions measured in the directions of increasing (Epa) and decreasing (Epc) potentials.

Crystallization

All crystallization experiments were done in an anaerobic glove box at <1 ppm O2 using degassed solutions. Y13C FdI is less soluble than native FdI so we were unable to induce nucleation using seed crystals of native FdI or other mutants as done previously (20, 21). Refinement of the native crystallization conditions (8, 9) did not yield crystals. Subsequently, crystallization screening was done using the vapor diffusion method and conditions developed by E. A. Stura (22). Clusters of small needle-like crystals were observed using sodium citrate as a precipitant. However, refinement of these conditions did not produce larger crystals, although the crystals could be grown reproducibly. On an attempt to induce nucleation in droplets that had not formed the small needle-like crystals after 18 days, several of the crystallization plates were transferred in sealed containers from the glove box at 27 °C to a room at 18 °C for 7 days, then to an incubator at 8 °C for 10 days, and then returned to the glove box. Temperature shifts are known to induce nucleation of the native protein (8). After five months (nine months total elapsed time) large, black rod shaped single crystals were observed in several of the drops.

Two crystals were mounted in sealed capillaries for data collection (Table I). Both crystals were grown from a 13 mg/ml protein solution in 25 mM Tris-HCl, pH 7.4, and in both cases the droplets consisted of 2 µl of protein solution mixed with 2 µl of a 1.0-ml reservoir solution. For the first crystal mounted, the reservoir solution was 1.26 M sodium citrate, pH 7.0, containing 0.5 mM beta -mercaptoethanol (beta ME). For the second crystal mounted, the reservoir solution was 1.32 M sodium citrate, pH 7.0, without beta ME added.

Table I. Data collection and refinement statistics


Crystal +beta ME  -beta ME

Size, mm 1.0 × 0.2 × 0.1 0.7 × 0.15 × 0.07 
No. of observations 25,133 10,648
No. of independent reflections 9,115 5,346
Rsymm (F), all data 0.067 0.114
I/sigma (I), all data 9.9 4.5
I/sigma (I), last shell 1.9 0.7
Resolution of last shell, Å 2.43-2.35 2.73-2.67
Completeness, all data 85.2% 73.3%
Completeness, last shell 32.9% 21.9%
R factor 0.216 0.203
Resolution, Å  8.0-2.35  8.0-2.67
Reflections >0sigma (F) 8577 4474
No. of protein atoms 1670 1670
No. of iron atoms 14 14
No. of sulfur atoms 18 18
No. of H2O molecules 0 0
Average B factor, Å2
  Molecule A 9.64 9.05
  Molecule B 9.63 9.61
Root-mean-square deviations from ideality
  Bonds, Å 0.016 0.017
  Angles, degree 3.55 3.88
  Planes, Å 1.49 1.81

Structure Determination

Diffraction data were collected using CuKalpha radiation from a Ru200 x-ray generator operated at 40 kV, 80 mA, and equipped with a graphite monochromator and Siemens area detector mounted on a four-circle goniostat. Data were collected by both phi  and omega  scans in increments of 0.25° with exposure times of 400 s/frame. To minimize the effect of decay, the first crystal was translated in the beam twice. The total time for data collection was 9 days for the first crystal (with beta ME) and 4 days for the second crystal (without beta ME). The data from both crystals were indexed, integrated, merged, and scaled using the Xengen suite of programs (Table I) (23). The space group of both crystals is P21 with a = 39.3, b = 73.7, c = 45.1A, and beta  = 98.4 ° and two molecules (A and B) of Y13C FdI (2.67 Å3/Da) per asymmetric unit.

The structure was solved by molecular replacement using the data set from the first crystal and the Xplor version 3.1 suite of programs (24). The native FdI structure from tetragonal crystals (8, 9) with residue 13 modeled as a glycine was used as a search model. Rigid body, positional, and isotropic B factor refinement of the structure representing the two best solutions to the rotation and translation searches resulted in an R factor of 0.23 for all data in the resolution range 8.0-2.5 Å. An unbiased 2|Fo|-|Fc| Fourier map computed with all data in the range 20.0-2.5 Å revealed 5sigma electron density at residue 13 in place of tyrosine of native FdI in both molecules A and B, indicative of cysteine persulfide. The electron density was characteristic of both a cysteine side chain and a disulfide (Fig. 2). A model for a cysteine persulfide residue (CSS) was constructed and fit to the density using the XtalView suite of programs (25). The model consists of a cysteine amino acid side chain (Calpha , Cbeta , Sgamma ) with an additional sulfur atom (Sdelta ) bonded at 2.02 Å to Sgamma ). The Cbeta -Sgamma -Sdelta bond angle is tetrahedral. The CSS residues were refined in Xplor using charge and energy restraints of a disulfide for the Sgamma -Sdelta bond and a protonated cysteine sulfur for the terminal Sdelta atom. The side chains of a total of 34 Glu, Asp, Lys, Arg, and Gln residues on molecules A and B were adjusted to fit the electron density in 2|Fo|-|Fc| maps due to changes in their conformation in the monoclinic crystal form relative to the tetragonal form. The final model was refined against all observed data to 2.35 Å (Table I). There are no outliers in the Ramachandran plot except for four glycines in both molecules A and B. Co-ordinates have been deposited with the Protein Data Bank.


Fig. 2. Electron density for the cysteine persulfide residue in Y13C FdI in molecule A (CSS13) and molecule B (CSS213) in crystals grown in the presence of beta ME (A and B) and absence of beta ME (C and D). The maps are calculated using coefficients 2|Fo|-|Fc| and the refined coordinates of Y13C FdI (Table I) and are contoured at 1, 2, 3, 4, and 5sigma . In A and B, all data in the resolution range 20.0-2.35 Å were used; in C and D, all data in the range 20.0-2.67 Å were used. Cbeta , Sgamma , and Sdelta correspond to CB, SG, and SD atoms, respectively.
[View Larger Version of this Image (90K GIF file)]

Because the first crystal was grown from a droplet containing beta ME, the possibility existed that the additional density observed on the cysteine introduced at position 13 was due to beta ME, if slow oxidation of the cysteine had occurred over the extended crystallization time. Although no additional density was observed for carbon and oxygen atoms of beta ME on either molecule A or B, it was possible that these atoms could have been disordered while sulfur bonded to Cys13 was not. Therefore, a second data set was collected from a crystal grown in the absence of beta ME (Table I). The data from this crystal was scaled to the first, and a |Fo|-|Fc| map was calculated using data in the range 20.0-3.0 Å and phases based on the refined model of Y13C FdI, including CSS residues on both molecules A and B. The difference Fourier map was featureless at residue 13, indicating no difference in the crystals grown without beta ME versus beta ME. Subsequently, a 2|Fo|-|Fc| map calculated with all data in the range 20.0-2.67 Å revealed a very similar density for the CSS residue on molecules A and B as observed in the first crystal (Fig. 2). Therefore, we conclude that beta ME is not a factor in the formation of monoclinic Y13C FdI crystals, and it is not present as a modification of Cys13. The Y13C FdI structure was also refined against all observed data to 2.67 Å (Table I).

Crystallographic Evidence for a Cysteine Persulfide

In addition to the shape and height of the electron density for both CSS13 and CSS213 in both the structure with and without beta ME added (Fig. 2), the presence of a cysteine persulfide was demonstrated by a series of refinement calculations in which each sulfur or oxygen of each non-ligand cysteine and serine was modeled as oxygen and sulfur, respectively (Table II). For example, Cys11 was modeled as "Ser11," and Ser56 was modeled as "Cys56." Similarly, the terminal atom on the Cys13 side chain was modeled as sulfur (the CSS residue) or oxygen, a possible Cys13-SOH residue. The only other free Cys or Ser in FdI is Cys24, which was also modeled as "Ser24." The corresponding residues in molecule B were modified in the same way. A series of refinement calculations were carried out using the 2.3 Å data from the crystal grown with beta ME (Table I), Xplor3.1, and the same charge and energy restraints used to refine the Y13C FdI structure (Table I). For the test Cys13-SOH side chain, the residue was defined to have a Sgamma -Odelta bond of 1.8 Å, tetrahedral angle for Cbeta -Sgamma -Odelta and OH group as in serine. The eight models, in which only one sulfur or oxygen atom at a time was changed, were independently refined. Three of these models with Ser11, Ser24, and Cys56 were also refined against the native FdI data (9) at the same resolution by the same protocol as a further control, since the native FdI crystallizes in a different space group. Finally, the correct native structure with Cys11, Cys24, and Ser56 was refined at 2.3 Å for comparison.

Table II. Refinement of sulfur versus oxygen at 2.3 Å resolution


Control structure
Test case
Residue Atom Electron density B Residue Atom Electron density B

 sigma Å2  sigma Å2
Y13C FdI data
  Cys11 Sgamma +5 3.1 Ser11 Ogamma +5 2.0
  CSS13 Sgamma +5 7.3 Cys13 Sgamma +5 6.9
  CSS13 Sdelta +4 12.0 Cys13 Odelta +4 2.0
  Cys24 Sgamma +5 8.6 Ser24 Ogamma +5 2.0
  Ser56 Ogamma +1.5 13.8 Cys56 Sgamma +1.5 25.6
  Cys211 Sgamma +5 4.5 Ser211 Ogamma +4 2.0
  CSS213 Sgamma +5 9.9 Cys213 Sgamma +4 5.7
  CSS213 Sdelta +4 13.6 Cys213 Odelta +4 2.0
  Cys224 Sgamma +5 4.4 Ser224 Ogamma +5 2.0
  Ser256 Ogamma +1.5 17.4 Cys256 Sgamma +1.5 27.2
Native FdI data
  Cys11 Sgamma +5 8.0 Ser11 Ogamma +5 2.0
  Cys24 Sgamma +5 7.9 Ser24 Ogamma +5 2.0
  Ser56 Ogamma +1.5 12.1 Cys56 Sgamma +1.5 22.8

Table II summarizes the results of the test refinement calculations. The height of the electron density of the atom in question in a 2|Fo|-|Fc| map, calculated in each case with all data in the range 20.0-2.3 Å, is given in intervals of 1sigma of the electron density, and the isotropic temperature factor (B factor) for each atom in question is given. In each and every case where a sulfur atom was modeled as an oxygen, the B factor refined to 2.0 Å2, the minimum allowed by the program, indicative that the model had insufficient electron density at that site. In particular, a model with oxygen bonded to Sgamma of Cys13 refines to 2.0 Å2 on both molecules A and B, while the Sgamma atom retains a B factor similar to that observed with a persulfide (CSS residue) as a model. Conversely, when an oxygen is modeled as a sulfur (Ser right-arrow "Cys") the B factor always becomes much larger, indicative of excess electron density at the site in the model. Thus, the fact that a Sdelta atom on CSS13 and CSS213 refines to a reasonable B factor indicates that it is not oxygen. Finally, the electron density in each and every case is 4-5sigma for sulfur, whether modeled as sulfur or oxygen, and 1.5sigma for oxygen in serine, whether modeled as sulfur or oxygen. Together, these data demonstrate that Cys and Ser can be discriminated at 2.3 Å resolution, and that the atom bonded to Sgamma of Cys13 is more massive than oxygen and has electron density comparable to that of sulfur in other cysteine residues with comparable B factors. In principle, this atom could be aluminum, silcon, phosphorus, chlorine, or argon in terms of x-ray scattering; however, none of these atoms are likely or able to bond singly to sulfur. Therefore, we conclude that the CSS residue is a cysteine persulfide.


RESULTS AND DISCUSSION

Ferredoxins are small acidic electron transfer proteins that contain [Fe-S] clusters attached to the polypeptide via cysteine residues. When a ferredoxin contains a [4Fe-4S]2+/+ cluster three of the four cysteine ligands are usually supplied by a highly conserved CysXXCysXXCys sequence motif (1, 6). Little is known about the in vivo assembly of these clusters and the role that the sequence motif plays in that process. Where x-ray structures are available it is clear that the clusters are buried (7-13), which suggests that they may be assembled during the protein folding process rather than after the protein is fully folded. For the simpler ferredoxins the protein can fold in vitro from apoprotein with the correct [4Fe-4S]2+/+ in place (26, 27), but in vivo other proteins are likely to be involved in the delivery of iron and sulfide (28). Whether the normal assembly process requires a specific sequence motif or just a specific three dimensional orientation of protein cysteine ligands is not known.

AvFdI is a 7Fe protein that contains a [3Fe-4S]+/0 cluster ligated by cysteinyl residues at positions 8, 16, and 49 (7-9). Although there is a free cysteine at position 11, that cysteine is far removed from the [3Fe-4S]+/0 cluster and could not be used as a fourth ligand to the cluster in the absence of substantial structural rearrangement. In this study the x-ray structures of AvFdI and PaFd (7-13) were used as a guide in attempts to obtain the correct three-dimensional orientation of four cysteines without creating a CysXXCysXXCys sequence motif.

Fig. 1 shows the structures of one of the CysXXCysXXCys loops of PaFd (11, 12), the corresponding Cys8XXCys11XTyr13XXCys16 loop from AvFdI (9), and a model for the Cys8XXCys11XCys13XXCys16 loop in a Y13C variant of AvFdI. The model was obtained by replacing the Tyr13 side chain with that of cysteine, without alteration of the main chain atoms, and rotating the chi 1 torsion angle from g+ to g- to illustrate the potential for proximity of the Cys13 Sgamma atom to the [3Fe-4S] cluster. The three models are shown in the same orientation to emphasize their overall similarity and to point out that in AvFdI the two residues between Cys8 and Cys11 represent a bulge expanding the upper half of the loop. Therefore, in structural terms Cys11 corresponds to residue 9 of PaFd, and Tyr13 corresponds to the Cys11 ligand to a [4Fe-4S] cluster in PaFd. If the two residue bulge in AvFd is deleted, and the remaining atoms superposed onto PaFd, the main chain Phi , Psi  angles of the two loops are seen to be very similar, as evidenced by a root-mean-square deviation of 31 pairs of corresponding atoms after least squares fit of 0.53 Å. Based on this observed structural similarity, it was anticipated that a Tyr213 to Cys replacement in AvFdI should result in a protein capable of forming a [4Fe-4S] cluster as in PaFd.

X-ray Structure Determination

As described under "Experimental Procedures" and shown in Table I, the x-ray crystal structures for two crystals of Y13C, one grown with beta ME and one grown without beta ME, were solved. Both crystals had two molecules (A and B) in the asymmetric unit. Fig. 2 shows the electron density for the refined Cys13 residue of Y13C in both molecules A and B in the crystallographic asymmetric unit in both a crystal grown with or without beta ME present. Two conclusions arise from analysis of these data. First, the engineered Cys13 fails to serve as a cluster ligand and second, it has been modified to become a cysteine persulfide. The structure of Y13C FdI refined against the higher resolution, more complete data set collected from a crystal grown in the presence of beta ME (Table I) will be used as a basis for further discussion. The geometry of the persulfides is normal and their conformations are very similar in molecule A and B: Sgamma -Sdelta bonds 2.00, 2.01 Å and Cbeta -Sgamma -Sdelta angles 104.4° and 108.8°, respectively. The B factors for the persulfides are also similar, being 4.3, 7.3, and 11.9 Å2 for Cbeta , Sgamma , and Sdelta respectively, on molecule A, and 6.9, 9.9, and 13.6 Å2 for these atoms on molecule B. Overall molecules A and B are very similar with an root-mean-square deviation for 106 Calpha atoms after least squares fit of 0.126 Å and a very similar distribution of average B factor per residue over the length of the polypeptide chain. The two copies of FdI in the asymmetric unit are also very similar to native FdI in tetragonal crystals (9) with rms deviations for 106 Calpha atoms after least squares fit of 0.179 and 0.183 Å. However, the conformations of several surface side chains, in particular Lys98, are rearranged due to the alternate crystal packing. In the monoclinic unit cell similar contacts occur between residues at the C terminus and the [4Fe-4S] cluster binding loop as in tetragonal crystals, while a local 2-fold axis relating molecule A and B is orthogonal to b, inclined 55° from a, but does not intersect the 21-screw axis. This interaction creates favorable contacts involving Lys10, Thr82, Glu83, Asp93, Gly96, Lys98, Gln102, and His103.

Fig. 3 shows the environment of the cysteine persulfide residue relative to Tyr13 in native FdI. The persulfide retains hydrophobic contacts to Cys11, Pro50, Pro87, and Ala91 present in native FdI for Tyr13; a hydrogen bond involving Tyr13 and Asp95 is lost. The van der Waals surface of the persulfide occupies a similar volume as the tyrosine, except that, by virtue of being a shorter side chain, the space filled by the tyrosine hydroxyl and Czeta atoms and some of the space filled by the Cepsilon 1 and Cepsilon 2 atoms, is vacant. Consequently, the [3Fe-4S] cluster remains shielded from solvent in Y13C FdI, but is closer to the solvent accessible surface of the protein. In effect, residue 13 in both structures fills a small cleft formed by the cysteine containing loops of residues 8-16 and 47-54 on either side of the [3Fe-4S] cluster (Fig. 3).


Fig. 3. Stereo figure of the protein environment of the CSS residue in the structure of Y13C FdI (thick lines) superimposed by least squares fit onto the structure of native FdI (thin lines). In this view the loop of the residues Cys8XXCys11X(13)XX Cys16 folds around the upper half of the [3Fe-4S] cluster while the loop of residues Pro47XCys49Pro50XXXIle54 folds around the lower half of the cluster. The short contact between Sgamma of CSS13 and an inorganic S of the [3Fe-4S] cluster is indicated. Note the proximity of Pro50 and Pro87 on either side of residue 13. The surface of the protein is toward the right. Molecule A of Y13C FdI is shown; molecule B is very similar.
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The occurrence of persulfides in protein structures is not without precedent. In the Q206C mutant of subtilisin, a persulfide is observed at the introduced cysteine (29). In this case, as well as in human superoxide dismutase (30), a persulfide apparently arises as an artifact of the purification procedure. In bovine liver rhodanese, a persulfide is observed at the catalytically essential Cys247 in the active site; the electron density of this residue at 2.5 Å resolution is very similar to that shown in Fig. 2 (31). In Desulfovibrio gigas ferredoxin II (FdII) additional electron density is observed on Cys11, a residue analogous to Cys11 in AvFdI (32). In D. gigas FdII Cys11 is not a ligand and a [3Fe-4S] cluster is present in the protein; the additional density was modeled and refined as methane thiol (32). In Y13C FdI there is no additional electron density beyond the Sdelta atoms of the persulfides. At this point it is not clear how the Y13C FdI persulfide arose but one possibility is that it was formed in vivo via the action of NifS or a NifS-type enzyme (33). These pyridoxal phosphate-containing enzymes appear to assist [Fe-S] cluster assembly by donating sulfide (33), possibly by formation of persulfides on ligand cysteines. It is interesting to note that neither of the free cysteines present in FdI is modified, whereas the engineered Cys13 is a persulfide. This suggests that if NifS is involved in modifying Cys13 there must be specificity to the reaction, either involving a Cys ligand motif or a so far unrecognized structural motif.

Fig. 4 shows the immediate environment of the CSS13 residue on molecule A of Y13C FdI. The structure of molecule B is virtually identical. Considering the three divalent, inorganic sulfur atoms of the [3Fe-4S] cluster, Sgamma of Cys11 and CSS13, there are six sulfur atoms in close proximity in this structure, and seven interatomic distances less than 6 Å. The conformation of Cys11 is very similar to that in native FdI. Sgamma of CSS13 has the most contacts (four), consistent with the expectation that, if residue 13 were a cysteine, then this sulfur atom could potentially be a ligand to a fourth iron atom, forming a [4Fe-4S] cluster. In particular, the contact between this Sgamma and S12 of the [3Fe-4S] cluster is short, 3.44 Å, or 0.26 Å less than the combined van der Waals radii of two sulfur atoms. This distance is comparable to those between S9, S11, and S12 (average, 3.47 Å) and within the range of S-S distances observed in other [3Fe-4S] and [4Fe-4S] clusters (32). For comparison, the contact between Cys24 Sgamma , a non-ligand cysteine, and inorganic sulfur of the [4Fe-4S] cluster in native FdI is 3.42 Å. Therefore, under oxidizing conditions it could be expected that a S-S bond could form between CSS13 Sgamma and S12, as indeed occurs between Cys24 and the [4Fe-4S] cluster in the presence of ferricyanide (34). The structure does not, however, explain the observation described below that Y13C FdI is unstable under reducing conditions.


Fig. 4. The immediate environment of the cysteine persulfide (CSS13) in molecule A of Y13C FdI. Interatomic distances involving sulfur atoms of CSS13, Cys11, and the [3Fe-4S] cluster less than 6 Å are indicated. In this view the loop containing Cys11 × CSS13XXCys16 wraps around the face of the [3Fe-4S] cluster containing three divalent, inorganic S2- atoms (S9, S11, S12). The cluster ligands Cys8, Cys16, and Cys49 are also shown. Other residues in contact with CSS13 are Pro50, Pro87, and Ala91.
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Absorbance and CD Spectra

The UV-visible absorption spectra of Y13C FdI are shown in Fig. 5 compared with those of native FdI. In the O2 oxidized state these spectra are indistinguishable. These spectra were also unchanged by the anaerobic addition of 2 mM dithiotheritol, which was added in an attempt to reduce the persulfide. Native FdI is an air-stable protein. Y13C is also fairly air stable when compared with some other FdI mutants (20), but it is not as stable as the native protein. For example, Y13C FdI could be stored at room temperature in air for about 24 h without cluster destruction, but longer storage eventually led to complete bleaching of the protein. Therefore the protein was purified anaerobically. As shown in Fig. 5 the addition of dithionite to either native or Y13C FdI resulted in partial bleaching of the spectrum. For the native protein this is known to arise from the reduction of the [3Fe-4S]+ cluster to the 0 oxidation state, because the reduction potential of the [4Fe-4S]2+ cluster is too low to be reduced by dithionite at this pH (35). The data in Fig. 5 show that the Y13C FdI dithionite reduction behavior is parallel to that of native FdI; however, we were surprised to observe that the Y13C FdI variant was extremely unstable in the dithionite-reduced state with complete destruction of both clusters occurring within 90 min of the addition of dithionite. Thus, although the protein was purified anaerobically, dithionite was not added to any of the buffers. It should be noted that the instability on reduction is not dependent upon the presence of dithionite as it is also observed in the direct eletrochemical experiments described below.


Fig. 5. Absorption spectra of native AvFdI (thin line) and Y13C FdI (thick line) in 0.1 M Tris-HCl, pH 7.4. Following 20 min of air oxidation and after reduction by bringing degassed samples to 2 mM in Na2S2O4; protein concentrations were ~30 µM.
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CD spectra of oxidized and reduced native FdI and Y13C FdI obtained at pH 7.4 are compared in Fig. 6. The wavelength dependence and form of the oxidized spectra are again extremely similar, strongly suggesting that the two proteins should have the same cluster composition. For the dithionite-reduced protein, the wavelength dependence is also similar but not identical. However, the amount of time required to prepare the samples and collect the data was sufficiently long that some cluster destruction may have occurred in the reduced state so that the minor differences may not be significant. To obtain more information about the reduced [3Fe-4S]0 cluster, we carried out the CD measurements at both high and low pH. The wavelength dependence and form of the CD spectra of native AvFd are quite different depending upon the pH due to the direct protonation of the reduced cluster at low pH (15, 36). The same changes in CD are observed for the Y13C FdI on going from high to low pH, indicating that its reduced [3Fe-4S]0 can also be directly protonated (data not shown). However, again the experiments must be carried out quickly, because the Y13C protein is extremely unstable in the reduced state.


Fig. 6. Visible region CD spectra of native AvFdI (thin lines) and Y13C FdI (thick line) in 0.1 M Tris-HCl, pH 7.4. A, following 20 min of air oxidation and B, after reduction by bringing degassed samples to 2 mM in Na2S2O4; protein concentrations were ~40 µM.
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Electrochemistry

Further confirmation of the similarities between the native and Y13C variant of FdI was obtained by electrochemical characterization. Despite the instability of the protein, it was possible to determine reduction potentials over a limited pH range using protein-film voltammetry in which a monolayer or less of sample molecules contacting different solutions can be examined in a short period of time (19). Fig. 7 shows base line-corrected voltammograms, measured at 20 mV s-1 for the direction of increasing potential, which reveal the general manner in which the different cluster redox transitions in the Y13C FdI vary with pH. The most prominent feature, by reference to previous studies (37), is assigned to the novel pH-dependent two-electron couple [3Fe-4S]0/2- and is designated C'. Likewise, the signal with the most positive potential is designated A' and is assigned to the well documented couple [3Fe-4S]1+/0, while the remaining signal (B') overlaid completely by C' at pH < 7.0 corresponds closely to the potential values of the couple [4Fe-4S]2+/1+ in the native protein.


Fig. 7. Voltammograms for Y13C AvFdI at various pH values obtained using a pyrolytic graphite edge electrode. The protein film is stabilized by co-adsorption of polymyxin. Scans are shown in the oxidative direction only and a polynomial baseline has been subtracted. The buffer-electrolyte solution contained 0.1 M NaC1, 20 mM mixed buffer (5 mM of acetate, Mes, Hepes, and TAPS) and 200 µg/ml polymyxin. Inset, dependence of reduction potentials on pH for Y13C AvFdI. Only one point is shown for couple B' due to interference by signal from couple C' at lower pH.
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Fig. 7 shows the pH dependence of reduction potentials determined from the average of the oxidative and reductive peak potentials. No differences were observed between values obtained at scan rates 20, 50, and 100 mV s-1. Notwithstanding the narrow range over which data were obtained, the curve for couple A' could be fit to a scheme consistent with protonation of the "0" oxidation level at pK = 7.2 ± 0.3, and E0' (alkaline form) = -417 ± 10 mV. The line for couple C' is very steep, and, overlaying couple B', it was not realistic to attempt analysis beyond linear regression, which gave a slope of -74.5 mV/pH unit. At pH 7.0, couples A', B', and C' have reduction potentials -402 ± 5, -640 ± 10, and -740 ± 10 mV, respectively, values previously obtained for the native protein. These data therefore indicate that the replacement of tyrosine by a cysteine persulfide does not affect either the pH dependence or reduction potential of the [3Fe-4S] cluster. Attempts were made to detect iron uptake into the [3Fe-4S]0 cluster using the procedure previously described (38) for observing cluster transformations in FdIII from Desulfovibrio africanus. No changes in signals were observed in experiments carried out at pH 7.0, with Fe(II) concentrations of 370 and 690 µM in the buffer electrolyte and holding the potential at -460 mV for various times before cycling.

EPR

In general, the presence of a [3Fe-4S]+ cluster in any protein is easily identified by the appearance of a characteristic g = 2.01 EPR signal (14). As shown in Fig. 8 the EPR spectrum of oxidized Y13C FdI was found to be both qualitatively and quantitatively indistinguishable from that of native FdI. As is the case for native FdI, Y13C FdI is EPR silent in the dithionite-reduced state, consistent with the formation of [3Fe-4S]0. No evidence for the conversion of that cluster to a [4Fe-4S]2+/+ cluster was obtained when the reduction was carried out in the presence of excess iron.


Fig. 8. EPR spectra of native AvFdI (thin line) and Y13C FdI (thick line) at 11 K following 20 min of air oxidation in 0.025 M Tris-HCl, pH 7.4. Protein concentrations were ~100 µM for native FdI and ~91 µM for Y13C FdI. The microwave power was 0.05 mW, the modulation amplitude was 5.1 G, and the microwave frequency was 9.43 GHz.
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Taken together these data demonstrate that, despite the introduction of an additional Cys residue in the correct structural location, a [4Fe-4S]2+/+ cluster fails to form in that location.

Why does Y13C FdI not form a [4Fe-4S]2+/+ cluster in the [3Fe-4S]+/0 location? One possibility is that a CysXXCysXXCys motif is actually required for the in vivo assembly of a [4Fe-4S] cluster in that location. Another is that the presence of the persulfide may prevent Cys13 from serving as a ligand. Alternatively it may be that the presence of the persulfide indicates that the assembly process was initiated by addition of a sulfide to Cys13, but that the subsequent use of that residue as a cluster ligand was somehow prevented. For example, the [3Fe-4S] cluster may already have had its full complement of inorganic sulfur and therefore have been unable to use CSS13 to insert an inorganic sulfur.

Analysis of the structure also provides clues as to why Cys13 might not be able to serve as a cluster ligand even if the persulfide could be cleaved in vivo. Thus, residue 13 and its neighbors are involved in a number of contacts with the surrounding protein that may restrain Cys13 from undergoing small but necessary conformational changes. That some adjustment is needed is indicated by the Sgamma to [3Fe-4S] cluster divalent sulfur distances which cannot be made symmetrical by simple rotation of the Cys13 chi 1 torsion angle. In the Y13C FdI structure these distances at best range from 2.5 to 4.2 Å, whereas in the PaFd structure where an iron atom of the [4Fe-4S] cluster is ligated, the corresponding distances are 3.5 to 4.1 Å. Therefore, even if the additional sulfur of the persulfide were removed Cys13 would have to shift ~1 Å to ligate Fe as Cys11 does in PaFd. In the structure of AvFdI (9) at least 11 hydrogen bonds may restrain such a shift; the carbonyls and amides of residues 11-15 are involved in hydrogen bonds with the main chain atoms of Cys8, Val17, Glu18, and Leu88, and with the side chains of Thr14 and Lys84; and the side chains of Lys12, Thr14, and Asp15 are involved in hydrogen bonds with the carbonyl of Gly28, amide of Lys85, and the side chains Glu27 and Lys84, forming two salt bridges. In previous studies we have shown that the [4Fe-4S]2+/+ cluster of AvFdI is able to switch one remote Cys ligand for another Cys residue (23, 38, 42). It should be noted, however, that in the structures of those C20A and C20S FdI variants considerable rearrangement takes place to accommodate a new ligand (Cys24) to a [4Fe-4S] cluster (20, 39). In those cases the loop undergoing the conformational change, Cys20XXXCys24, is on the surface of the protein and is involved in only two hydrogen bonds with other residues.

Physiological Consideration

The data just described demonstrate that Y13C FdI is extremely similar to native FdI in its cluster organization and redox properties, but that it differs from native FdI in being unstable in the reduced state and in the presence of oxygen. The protein also accumulates to only about 5% of wild-type levels in vivo, which may reflect either an initial folding problem or increased oxygen sensitivity leading to protein destruction. In native FdI, Tyr13 is partially exposed on the surface of the protein but also is in contact with the [3Fe-4S] cluster (9). As indicated above, the replacement of this large, aromatic residue with a cysteine persulfide creates a cavity adjacent to the cluster. Although the cluster is not directly exposed to solvent, the smaller CSS13 chain is unlikely to be as effective in shielding the cluster, perhaps explaining the increased oxygen sensitivity of this mutant and its instability in the reduced state. The Tyr13 side chain also has hydrophobic contacts with Cys11, Pro50, Pro87, and Ala91, and is involved in a hydrogen bond with Asp95. The loss of these contacts may expose additional hydrophobic surface area in Y13C FdI as evidenced by the reduced solubility of the mutant protein at high ionic strength, as observed in our early unsuccessful crystallization trials. The exposure of this additional hydrophobic surface might also contribute to the lowered stability of the Y13C FdI variant.

In previous studies we have shown that AvFdI has an electron-transfer function that is important for cell growth (35, 40) and a regulatory function in controlling the expression of the fpr gene product (40-42). Y13C FdI appears to be able to carry out both of these functions as evidenced by the normal growth of the Y13C overproduction strain and by the observation that FPR levels are the same in strains overproducing the native and Y13C variants of FdI. If the in vitro instability of the reduced protein discussed above is due to increased solvent exposure, then something appears to prevent this exposure from becoming a problem in vivo.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant GM-45209 (to B. K. B.), GM-36325 and GM48495 to (C. D. Stout) and by grants from UKEPSRC and BBSRC (to F. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Atomic coordinates and structure factors (Code 1FTC) were deposited in the Protein Data Bank, Brookhaven National Laboratory.


par    Current address: City of Hope, 1500 E. Duarte Rd., Duarte, CA 91010.
**   To whom correspondence should be addressed. Tel.: 714-824-4297; Fax: 714-824-8551; E-mail: bburgess{at}uci.edu.
1   The abbreviations used are: Fd, ferredoxin; AvFdI, Azotobacter vinelandii ferredoxin I; PaFd, Peptococcus aerogenes ferredoxin; beta ME, beta -mercaptoethanol; CSS, cysteine persulfide residue; PIPES, 1,4-piperazinediethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid.

ACKNOWLEDGEMENTS

We thank E. A. Stura for invaluable discussions and Hayley Angove for assistance with EPR spectroscopy.


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