7-iron ferredoxin revisited*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

7-Iron Ferredoxin Revisited*

Vol. 263, No. 19, Issue of July 5, pp. 9256-9260,1988 Printed in U. S. A.

(Received for publication, March 14, 1988)

Charles David Stout From the Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037

The crystal structure of the 7Fe ferredoxin from Azotobacter vinelandii has been redetermined using area detector data to 2.7-A resolution and a new derivative. Tetragonal crystals of the protein were main- tained at pH 8.0. The results show that the structure previously reported was in error and confirms a recent independent report of the structure (Stout, G. H., Tur- ley, S., Sieker, L. C., and Jensen, L. H. (1988) Proc. Natl. Acad. Sei. U. S. A. 86, in press). The protein fold is similar to the homologous 8Fe ferredoxin structure for the N-terminal half of the protein; the C-terminal residues wrap around this structure. The structure contains a 3Fe cluster coordinated by cysteines 8, 16, and 49 and a 4Fe cluster coordinated by cysteines 20, 39, 42, and 46. However, there are two free sulfhydryls, cysteines 11 and 24, in the new model. Cysteine 24 is in contact with the [4Fe-4S] cluster. Cysteine 11 is shielded from solvent by residues 86-90.

Azotobacter uinelandii produces a 7Fe ferredoxin which was first characterized in 1970 (1). Two crystal forms were reported (2), and a 4.0-A resolution electron density map indicated that the protein contained two different Fe-S clusters, which were modeled as 4Fe and 2Fe centers (3). At 2.5-A resolution it was apparent that the smaller cluster contained three iron atoms (4) as independently demonstrated by Moss- bauer data (5). The cluster was modeled as a [3Fe-3S] cluster with five cysteine ligands, based on the sequence (6) and a chain trace for the protein (7). This model was subjected to refinement (8).

An independent investigation has indicated recently that the novel cluster present is a [3Fe-4S] cluster and that the protein is folded not as proposed (7), but like 8Fe ferredoxin (9). In order to resolve this issue, I have redetermined the crystal structure using new area detector data and a new heavy atom derivative. The results show that the previous analysis was in error. The new electron density map confirms the recently reported new chain folding and presence of a [3Fe-4S] cluster (9). However, the new model raises questions concerning the presence of two free cysteines and the role of the additional polypeptide in the structure. In addition, the pH of the crystals used in the two studies differs markedly, 6.5 (9) and 8.0 (this work).

EXPERIMENTAL PROCEDURES

Crystals were grown as described previously with Tris. HC1 buffer at pH 8.0 and 9 mg/ml protein, which provides a better yield of large single crystals (2). The crystals are tetragonal with unit cell constants

* This research is supported by National Institutes of Health Grant GM-36325. This is Publication 5205MB of the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

a = b = 55.2 A, c = 95.2 A, and one molecule (12,500 daltons) per asymmetric unit. Crystals of this form and a triclinic form were reproducibly grown using protein purified from both butanol extracts (2) and nitrogenase extracts (10). Dissolved crystals have absorbance ratios A ~ / A ~ w = 1.64 and Am/Azeo = 1.27. Diffraction patterns of proteins derived by either method were identical. However, the diffraction pattern of the tetragonal form shows non-isomorphous in- tensity differences and small changes in cell constants at more acidic pH. Film data seta to 2.7-A resolution for native tetragonal crystals equilibrated at pH 8.0 and 5.0 scale together with RF = 0.30. Crystals were equilibrated in a synthetic mother liquor of 0.15 M Tris.HC1, pH 8.0, in 85% saturated (NH&SO4 at room temperature for data collection.

The KZPtC1, derivative was made as described previously (4). A new GdZ(SO& derivative was prepared by soaking crystals in a solution in which the heavy atom salt replaced some of the (NH4),S04 of the stabilizing solution: 79% saturated (NH&SO,, 0.15 M Tris. HCl, pH 8.0, and 0.1 M Gd2(S04)3. Although not all of the salts dissolved, the ionic strength was sufficient to stabilize the crystals, which were soaked for 11 days at room temperature. The previously reported osmium and rhodium derivatives (7) were not prepared because of the subsequently observed effects of acidic pH on the intensities.

Data were collected with a Xentronics area detector and GX-21 rotating anode x-ray generator (nickel-filtered Cu/Ka, 40 kV, 100 mA, 0.5-mm focal spot). Twelve data sets were collected; two for the native, one each for the platinum and gadolinium derivatives, and eight as surveys for additional derivatives. The data were integrated, reduced, merged, and scaled using the Genex suite of programs (11). On average a data pet consisted of 20,000 observations of 4,300 unique reflections to 2.7 A, of which 10% were rejected. Data were scaled in point group 4/mmm, but rejected in 422 to preserve large Bijvoet differences. R.,,(F) values ranged from 5.5 to 8.0% in 4/mmm and from 3.8 to 6.0% in 422 for the 12 data sets. Values of <I>/<a(Z)> were typically 20.0-30.0 for an entire data set, and 4.0-6.0 at 3.0 A for derivatives or 2.7 A for native crystals. On average, R.,,(F) was 1.6% greater when Bijvoet pairs were averaged.

Phases were refined by the method of Wang (12) using the single major Pt site in both P41212 and P43212. Bijvoet difference Fourier maps (13) of the native data were calculated as described previously (3). The following was observed. For platinum at +x,+y,+z in P41212 (“++” phases), the map showed large positive peaks at the two Fe-S cluster sites. For platinum at -x,-y,-z in P43212 (“--” phases) the map showed large negative peaks at two sites related by inversion (1/ 2 - z ) from those in the ++ map, as predicted (13). As before (3), the maps calculated with the other two possible phase sets, +- and -+ showed much lower contrast and are incorrect assignments of the platinum position.

The results for the ++ and -- maps are inverted from those originally reported (3). This can occur if the signs of coefficients in the Bijvoet difference Fourier are reversed; i.e. the ++ map with negative coefficients has negative peaks at the correct sites, indicating the inverted (but incorrect) solution. An error reversing (F+) and ( F - ) must have occurred in the original analysis (3), and this must have arisen in the calculations, because data collected in a right- handed system from crystals in 422 (or 222) will preserve the signs of the Bijvoet differences regardless of crystal orientation (14). In- cluding the platinum and iron anomalous data in the phase calculations (15) for the derivative and native crystals gives a combined figure of merit of 0.54 for the ++ solution and 0.53 for the -- solution, indicating the P41212 space group. Furthermore, an isomorphous difference Fourier for the gadolinium derivative has a large positive peak at the gadolinium 1 site with ++ phases in P41212, but

9256

7-Iron Ferredoxin Revisited 9257

TABLE I Heavy atom parameters

Derivative Site A" x y z B (A2) Rb F"/E' Platinum 1 0.0188 0.4746 0.1389 0.1940 23.8 0.61 0.87

2 0.0043 0.2226 0.0496 0.1015 19.1 Gadolinium 1 0.0092 0.1097 0.4481 0.2429 25.0 0.69 0.88

2 0.0100 0.4632 0.0860 0.3564 23.5 3 0.0047 0.4724 0.3039 0.0049 15.7 4 0.0046 0.1658 0.0866 0.0473 17.1

Iron <4Fe> 0.1535 0.2737 0.3884 0.1198 15.7 0.35 <3Fe> 0.1419 0.3057 0.2909 0.2305 18.5

a Relative occupancy based on platinum scattering factor for platinum, gadolinium derivatives, and iron anomalous scattering factor for native data.

* R value for centric reflections for derivatives, and largest 35% acentric differences for native data.

Ratio of average heavy atom structure factor to lack of closure for all data to 3.0-A resolution.

no positive peaks whatsoever with -- phases in P43212 (15). Phases based on the platinum derivative and the iron anomalous

differences were applied to the derivative data sets, revealing four gadolinium sites and a minor platinum site. These sites were refined against the origin removed Patterson maps (16) a t 3.0-A resolution (Table I). Difference Fourier maps of the other eight data sets revealed only minor sites or no binding by heavy atoms. The redundant data sets were used to improve the iron anomalous phases. Each data set was filtered for the largest Bijvoet differences; reflections having both IF'I and IF-1 > 3 . 0 ~ ~ and I A F I > u(AF) were accepted, yielding about 35% of the differences in each data set. The two Fe-S cluster sites, along with heavy atom sites when present, were refined against the anomalous differences of each data set (17). R values ranged from 0.33 to 0.38. Phase probabilities were calculated to 2.7 A with the 11 filtered data sets and merged. The merged anomalous phase probabilities were then merged with the platinum and gadolinium 3.0-A resolution isomorphous phases, and the combined pbases refined (12) to a figure of merit 0.71 for 4347 reflections at 2.7-A resolution.

The electron density map was interpreted without superposition of the structure of 8Fe ferredoxin (18). Ca coordinates measured from a mini-map were used for building the entire sequence with the programs GRINCH (19) and FRODO (20). The model built into the experimental density map has R = 51% for 856 atoms with overall B = 20 A* and 4347 reflections to 2.7 A. One cycle of molecular dynamics refinement of this model with the program XPLOR (21) (2000 K, 0.5 ps) reduced R to 33% for all 4347 data with an overall B = 20 A' and no added solvent. Coordinates of this partially refined model have been deposited with the Protein Data Bank' to update and correct the previous set of coordinates.

RESULTS

Figures of the 7Fe ferredoxin model for Ca, cysteines, and Fe-S clusters are shown in Fig. 1. The protein can be described as a sandwich of @-strands enclosing the Fe-S clusters. Al- though not extensively hydrogen-bonded, the structural motif is of a @-barrel with amphophilic a-helices on the surface. In this model the hydrophobic side chains are packed in the interior, hydrophilic ones lie at the surface, and prolines and glycines are associated with turns. In other words, the model describes a globular protein obeying principles of protein structure, unlike the previously reported model for the structure (7). Importantly, the new model is folded like the 8Fe ferredoxin (18). The two proteins are strongly homologous for residues 1-50, except for two 2-residue insertions in the 7Fe protein (6). As in the 8Fe ferredoxin there is local 2-fold symmetry relating the two clusters, their cysteine binding loops, and reverse turns. Therefore, this structure agrees with the recently reported independent study of the 7Fe ferredoxin structure (9).

Coordinates submitted 1/20/88 to F. C. Bernstein, Protein Data Bank, Chemistry Dept., Brookhaven National Laboratory, Upton, NY 11973.

FIG. 1. Perspective (a) and stereo (b) views of the 7Fe ferredoxin structure showing main-chain atoms, cysteine side chains, and Fe-S clusters.

Electron density for the Fe-S clusters is shown in Fig. 2. The 3Fe cluster density is adequately accounted for with a [3Fe-4S] model with three attachments to the protein; in the structure these are cysteines 8, 16, and 49. The model used is the same as that which also fits into the electron density for the Fe-S cluster of inactive aconitase;' this model is a [4Fe- 4S] (S,), cluster Yith one iron and cysteine S, missing (24). However, at 2.7-A resolution this fit to the density does not prove that there must be four inorganic sulfurs in the cluster. The electron density suggests a single trivalent sulfur, where it is bulged, and three divalent sulfurs, where the density is dimpled due to the missing iron. The electron density for the [4Fe-4S] cluster shows the expected four tetrahedrally oriented connections to the protein, which are cysteines 20, 39, 42, and 45.

7Fe ferredoxin differs from ita 8Fe counterpart by having an additional cysteine, insertions in the N-terminal, cluster binding sequences, and an additional 52 amino acids at the C terminus. The additional protein is associated with novel structural features at each Fe-S cluster.

The loop containing the [3Fe-4S] cluster ligands is in contact with residues from the lengthened C terminus. The loop CysS-Ile-Lys-Cys"-Lys-Tyr-Thr-Asp-Cys16 coordinates the cluster at Cys' and Cys", but surprisingly not at Cys'l although this residue is adjacent to the cluster. The orienta-

* A. H. Robbins and C. D. Stout, unpublished results.


a

b

e,

D.

lcA

FIG. 2. Electron density of the Fe-S clusters. a, [4Fe-4S] cluster with four cysteine S, ligands. The density behind and to the right of the cluster in this view has been assigned to cysteine 24. b, [3Fe-4S] cluster with three cysteine S, ligands. The face of the cluster containing three divalent inorganic sulfurs, but missing a fourth iron site, is oriented toward lysine 12 and cysteine 11, the chain of density to the right.

tion of the ligands Cys', Cysl', and Cys4' is such that, if Cys" were to be a ligand, it could occupy the fourth position of a normal tetrahedral [4Fe-4S] cluster, but it does not. A reason for this may be the expanded loop Cys'l-Lys-Tyr-Thr-Asp- Cys''. In 8Fe ferredoxin this loop contains 2, not 4, residues between cysteines. The Ca of Cys" is displaced -4 A from a position opposite the vacant iron site; this position is occupied by Lys". The electron density for the Cys" side chain is not resolved from the main chain; however, the Ca position is fixed by side chain assignments for the other residues. The extended stretch of residues Cy~"-Lys'~-Tyr'~ runs antipar- allel to, and is in contact with, the sequence Asp=-Pro-Leu- Pro-Aspw of the C-terminal residues (Fig. 3a). These contacts,

forming an additional shell of protein around Cys", may constrain it from becoming a ligand. Other residues adjacent to the [3Fe-4S] cluster are Val4, Tyr13, Thr14, Val", Leu3', Ala51, Alab3, and Iles4.

While the additional protein in the 7Fe ferredoxin shields the [3Fe-4S] cluster, the [4Fe-4S] cluster is as exposed to solvent as in 8Fe ferredoxin. The ligands to the cluster, Cyszo, Cys3', and Cys4', are homologous with ligands in the 8Fe protein. The anti-parallel loop between residues 25 and 34 and the helical-like turn of residues 35-38 are similar, and the length of the linking peptide Cys'" to Cyszo is the same in both proteins. However, in 7Fe ferredoxin residues 26-30 are in contact with the C-terminal residues from 79 to 84 and 103


b

C

FIG. 3. Electron density of the protein. a, residues 86-93: Asp-Pro-Leu-Pro-Asp-Ala-Glu-Asp. Connecting density for Lys= is at upper left; that for TrpD4 is at lower left. Density to the left of this chain belongs to residues 11-13 and Prom. b, residues 20-25: Cys-Pro-Val-Asp-Cys-Phe. Density for the [4Fe-4S] cluster is shown without the model. c, residues 26-33: Tyr-Glu-Gly-Pro-Asn-Phe-Leu-Val. Density for PheZ5 is at upper left; that for Ile3' at upper right.

9260 ?"Iron Ferredoxin Revisited

to 105. A 2-residue insertion has an interesting effect on the structure at residues CysZ0-Pro-Val-Asp-Cysz4 (Fig. 3b). CysZo is a ligand to the [4Fe-4S] cluster, and ProZ1-Valz2 makes a reverse turn so that Aspz3 is oriented toward the solvent. Surprisingly, the side chain of CysZ4 is oriented directly toward the cluster. In the map, the farthest the S, atom can be placed from inorganic sulfur of the cluster and still fit the main- chain electron density is -3.3 A. As the electron density for all the residues is in register with the sequence (Fig. 3c), there appears to be no choice but to place the side chain of CysZ4 in contact with the [4Fe-4S] cluster. The side chains of Phe', Val", Proz1, Valz2, PheZB, Ile34, Ile4', and Leu44 are also adjacent to the cluster. As S , of Cys" is on the distal side of the 3Fe cluster from CysZ4, it seems unlikely that the two sulfhydryls could come into contact.

DISCUSSION

It is important and interesting to consider how the first reported structure for 7Fe ferredoxin (7) was in error. While the original enantiomer assignment was incorrect, the tech- nical error went undetected due to two subjective factors: the appearance of protein electron density maps, and the presence of a novel cluster. The map interpreted was essentially a SIR map based on the platinum derivative; the rhodium and osmium derivatives did not contribute significantly to the phases (7). Nevertheless, a chain trace appeared convincing given the known presence of a new structural feature, the 3Fe center, and inexperience in recognizing protein secondary structural features at 3.0 8, resolution. The starting model in P4&2 had a R value of 56% and refined to 35% (8) because the positions of the Fe-S clusters were correct with respect to this enantiomer, because the phases of the centric reflections (approximately one-fourth of the data) were correct and because some chain segments remain in density if the enantiomer is changed. For instance, comparison of the new and inverted, old models shows that superposition of residues 35- 50, which have similar conformations, results in alignment of residues 11-20 of the new model on residues 15-24 of the old model. Once refined, the incorrect model biased the phases so that even residue-deleted maps returned the false structure. Abnormal characteristics of the protein refinement have subsequently been pointed out (9).

The new model for 7Fe ferredoxin contains a [3Fe-4S] cluster with three cysteine ligands from the protein, one to each iron, as originally proposed for ferricycinide-treated 3Fe ferredoxin (24). A cluster model derived from a standard [4Fe- 451 cluster fits the density, although at 2.7-A resolution it cannot be proven that the cluster contains four inorganic sulfides. The previous model for a [3Fe-3S] cluster with Fe- Fe distances of -4 A arose from constraints, and subsequent bias, ef the incorrect chain trace (8). A [3Fe-4S] cluster with -2.7-A Fe-Fe distances is in agreement with EXAFS results for the 3Fe form of Azotobacter ferredoxin (24), for 3Fe Desulfovibrio gigas ferredoxin (25), and for inactive aconitase (26). Resonance Raman spectra of Azotobacter ferredoxin are interpretable with a cubane-like model for a [3Fe-4S] cluster (27). The presence of four inorganic sulfurs is consistent with the stoichiometry of the 3Fe cluster in inactive aconitase (26).

The free sulfhydryl of cysteine 11 is shielded from solvent by residues 86-90 and that of cysteine 24 appears to be in contact with the [4Fe-4S] cluster. This may explain the unreactivity of the cysteines to heavy atom reagents. Inter- actions of residues 86-90 with the chain near Cys" may also be the reason why the 3Fe cluster does not readily convert to

a 4Fe cluster (28). The near approach of the CysZ4 sulfhydryl to inorganic sulfur of the [4Fe-4S] cluster may account for the formation of a new EPR signal upon mild oxidation with ferricyanide (22).

The magnetic circular dichroism spectrum of the reduced 3Fe center in Azotobacter chroococcum 7Fe ferredoxin changes between pH 6.3 and 8.3 (23). Tetragonal crystals of oxidized A. uinelundii ferredoxin exhibit marked changes in intensities of high angle reflections between pH 5.0 and 8.0. While these effects may be unrelated, it will be of interest to compare the structures of Azotobacter ferredoxin at pH 8.0 and 6.5 (9). A native data set at pH 8.0 to 1.9-A resolution has been collected on the area detector (140,000 observations of 12,000 independent reflections, two crystals, 1.0 x 1.0 X 0.5 mm, at 2 "C).

Acknowledgments-I am grateful to A. H. Robbins and D. E. McRee for helpful discussions, to B. K. Burgess for samples of the protein, and S. A. Collett and M. Pique for graphics.

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7-iron ferredoxin revisited*

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