Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1·7 Å

J. Mol. Biol. (1991) 219, 693-715

Refined Crystal Structure of Ferredoxin Desulfovibrio gigas at l-7 A

II from

Charles R. Kissinger?, Larry C. Sieker, Elinor T. AdmanS and Lyle H. Jensen Department of Biological Structure SM20 of Washington, Seattle, WA 98195, U.S.A.

University

(Received

14 December

1990; accepted 4 March

1991)

The crystal structure of ferredoxin II from Desulfovibrio gigm has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 9157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the F&S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55. Keywords: ferredoxin; three-iron; Fe-S cluster; crystal structure; Desulfovibrio

1. Introduction Ferredoxin from the sulfate-reducing bacterium, Desulfovibrio gigas, is unusual in that the molecule exists in two oligomeric forms that contain different iron-sulfur clusters (Bruschi et al., 1976; Huynh et al., 1980). D. gigas ferredoxin I (DgFdI$) is a dimer (J. J. G. Moura, personal communication) in which each of the identical 6400 M, subunits contains a single 4F&S cluster. Ferredoxin II (DgFdII) is a tetramer of the same subunit in which each subunit contains a 3Fe4S cluster. The two forms can be interconverted reversibly (Moura et al., 1982). The reduction potential of DgFdI is -455 mV, while that of DgFdII is - 130 mV (Cammack et al., 1977). DgFdI has been shown to be more efficient than DgFdII in coupling pyruvate dehydrogenase t Present address: Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, School of Medicine, 725 N Wolfe Street, Johns Hopkins University, Baltimore, MD21205, U.S.A. $ Author to whom all correspondence should be addressed. Q Abbreviations used: DgFd, D. g@as ferredoxin;FFT, fast Fourier transform; r.m.s., root-mean-square; PaFd, Peptococcus aerogenes ferredoxin.

693 0022-2836/91/120693-23

$03.00/O

activity to H, evolution in D. gigas cell extracts to which pyruvate and purified hydrogenase have been added (Moura et al., 1978). Conversely, DgFdII is more efficient in coupling H, consumption via hydrogenase to the reduction of sulfite (Bruschi et al., 1976). DgFdII becomes active in the former reaction only after a lag phase, concurrent with the disappearance of the electron paramagnetic resonance signal indicative of the three-iron cluster form and the rise of that of the four-iron form (Moura et al., 1984). These observations suggest that the two forms of D. gigas ferredoxin have physiological significance and that DgFdII is not merely an artifact of isolation, as might be the case for some other proteins found to contain three-iron clusters (Thomson et al., 1981; Kent et al., 1982; Beinert & Thomson, 1983). This apparent conversion of cluster type when added to D. gigas cell extracts raises the possibility that such conversion could occur in vivo as a physiological control mechanism. D. gigas ferredoxin is a member of a large family of homologous bacterial ferredoxins (Fig. 1). Indeed, its structure was predicted to fold in a manner similar to that of the two-center four-iron Peptococcus aerogenesferredoxin, and to the single center four-iron Bacillus thermoproteol$icus ferre0

1991

Academic

Press

Limited

694

C. R. Kissinger et al. Dew ffowibr io gigas F&#xvccus aerogenes BmMus tkmnproteo&ticus DesuhlMBrio b-/a&s Norway Dewltkbrio africarws I Cfostridium tbermoaceticum Azotobacter vioelandii Desulfovibrio gigas Peptococcus aerogenes Bacillus thermqwoteo&ticus Desuifovibrio bacalatus Norway Desuifovibrio africanus I Ciostridium therm~eticum Azotobact~ vhmfandii

--PIE-V-NDDCMAC-E--ACVEICP-DVFEMNEEG --AYV-I-NDSCIAC-G--ACKPECPVNCIQQG--PKYTI-VDKESCIAC-G--ACGAAAP-OIYDYDYDEDG --TIV-IDHEECIGC-E--SCVELCP-EVFAMIDQE ARKFY-VDQDECIAC-E--SCVEIAP-GAFAMDPEI -MKVT-VDQDLCIAC-G--TCIDLCP-SVFDWDDE--AFV-V-TDNCIKC-KYTDCVEVCPVDCFYEGP--

I

I

DKAVV-INPDSDLDCVE--EAIDSCPAEAIVRS--SI-YA-IDADSCIDC-G--SCASVCPVGAPNPED-IAYVT-LDSDILIDD-M-MDAFEGCPTDSIKVADO EKAMV-TAPDSTAECAG--DAIDACPVEAISKE--EKAYVKDVEGASQEEVE--EAMDTCPVQSIEE----GLSHVIVREDSCAR--E--S-VNECPTEAKIKEV-NF-LV-IHPDECIDC-A--LCEPECPAQAIFSEDEV%

$=DDNQGIVEVP@=EPFDGDPNKFE #=EVPEGA %=MQEFIQLNAELAEVWPNITEKKDPLPDAEDWDGVKGKLQHLEF!

Figure 1. Amino acid sequences of some bacterial ferredoxins. The sequences were split so as to align each half of the molecule. Highly conserved residues are in bold type. Adapted from Rruschi & Guerlesquin (1988). The sequence alignment is that of those authors except that previously undeterminedresiduesfound crystallographically (seethe text) have beenaddedto the sequences of P. aerogenes and D. gigasferredoxins.

doxin (Fukuyama et al., 1988). A recent review (Beinert, 1990) summarizes recent developments in ferredoxins and other iron-sulfur proteins. Bacterial ferredoxins are thought to have evolved from a single ancestor after gene duplication (Eck & Dayhoff, 1966; George et al., 1985) and typically bind two Fe-S clusters. Ferredoxins that bind a single Fe-S cluster, such as that from D. gigas, appear to have evolved subsequently through lossof cysteine ligands in the C-terminal half of the molecule. D. gigas ferredoxin has the N-terminal Cys-X-X-Cys-X-X-Cys-X-X-X-Cys-Pro sequence typical of the bacterial ferredoxins that bind two Fe-S clusters, but it contains only two cysteine residues in the C-terminal half of the molecule. Cysteine residues 8, 11, 14 and 50 correspond to residues that bind one of the two Fe-S clusters in P. aerogenes ferredoxin (Adman et al., 1973), and have been presumed to bind the four-iron form of the single Fe-S cluster in D. gigas ferredoxin. Cysteine residues 18 and 42 correspond to two of the residues that bind the second Fe-S cluster of P. aerogenes ferredoxin. The assumed proximity of these two cysteine residues in D. gigas ferredoxin led to the suggestion that they might form a disulfide bridge (Bruschi et al., 1976). D. gigas ferredoxin presents a unique opportunity to study an iron-sulfur protein that readily accommodates either a three-iron or four-iron cluster. A knowledge of the three-dimensional structures of the two forms of D. gigas ferredoxin should shed light on the reasons for the differences in oligomerization, redox potential, and physiological activity in the two molecules. The more stable of the two forms, DgFdII, has been crystallized in this laboratory (Sieker et aZ., 1984). The crystallographic structure determination of DgFdII has been undertaken to establish the geometry of the 3Fe-4S cluster in the molecule at high resolution, to identify structural features that might facilitate the interconversion between the two Fe-S clusters in the two forms of D. gigas ferredoxin and to allow modeling

of the electron-transfer complex between DgFdI I and D. gigas cytochrome cs, also under study in this laboratory (Kissinger & Sieker, 1987). Presented here are the results of the structure determination and refinement at 1.7 A resolution and comparison with previously determined bacterial ferredoxin structures. The resolution attained from the data permits refinement of the positions of the iron-sulfur cluster atoms without stereochemica1 restraints. Preliminary accounts of this work have been published (Kissinger et al., 1988, 1989). 2. Experimental (a) Crystallization

Methods

and data collection

Purified DgFdII was supplied by ,J. ,J. G. Moura. 1. Moura and ,J. LeGall. Crystals were grown from 25 M-ammonium sulfate at pH 5.0 (Sieker et al.. 1984). The crystals are monoclinic, space group C2. a = 4087 A. b = 4528 A, c = 2647 A (1 A = 91 nm) and fl= 1047”. The size of the unit cell indicated 1 ferredoxin monomer per asymmetric unit and a volume per dalton of protein of 1.83 A3. Solvent thus occupies approximately 32% of the crystal volume (Matthews, 1968). Diffraction data to 1.7 A resolution were collected on a Krisel Control modified Picker FACS-1 diffractometer at room temperature (19” to 21”) using Ni-filtered CuKcc radiation. To minimize the effectsof radiation damageon the observed reflection intensities, data were collected from 3 crystals in overlapping resolution ranges. Table I is a summary of data collection statistics for the 3 crystals. Data from the 1st 2 crystals were collected using the w-20 scan technique with a scan rate of Z”/min and scan width of 1.4”. Data from the 3rd crystal were collected using the m-26’ step-scan technique (Hanson pt al., 1979) with 7 steps in 28 (0.1”) and a counting time 01 5 s/step. Friedel pairs were collected for all reflections. For each crystal. several reflections covering a broad resolution range were recollected every 8 h to allow correction for radiation damage as a function of time and diffraction angle. The maximum deterioration correction factor applied was 1.70. Empirical absorption corrections were also applied using the method of North et al. (1968). The maximum absorption correction factor applied was 1.89.

Crystal Structure

To minimize errors between Friedel pairs due to radiation damage, Friedel pairs were collected in groups of 20 at positive and negative 26. Local scaling (Matthews, & Czerwinski, 1975) was applied to all Friedel pairs before data from different crystals were merged. After local scaling, data sets were placed on a common scale using 250 reflections (Friedel pairs unmerged) common to crystals A and B and 1382 reflections common to crystals B and C. The R(F),,,srge was 9056 for crystals A and B, and 0059 for B and C. The final merged data set consisted of 4901 Friedel pairs and 279 centric reflections with intensities greater than zero, representing approximately 94 O/o of the possible reflections to 1.7 A. Fig. 2 shows, as a function of resolution, the fraction of reflections observed at 2 and 4 times their estimated standard deviation (or). An overall B factor of 12 AZ was calculated from a Wilson (1942) plot. (b) Structure

solution

by resolved

anomalous

phasing

We made numerous attempts to obtain heavy-atom derivatives for this ferredoxin, all to no avail. From our earliest efforts at solving the structure, we were aware that we had a substantial anomalous signal that we hoped to utilize in solving the structure. We made a conscious decision not to use the rotation function with a model of P. aerogenes ferredoxin to solve the structure, both to keep the resultant model unbiased, and to test the utility of anomalous scattering in the structure solution. The programs of Hendrickson et al. (1985) were used for the resolved anomalous phasing procedure. Before the phasing procedure was attempted, the Friedel pairs were again locally scaled, this time using the method of Smith & Hendrickson (1982). The data were divided into 3 resolution ranges and further divided into reflections with a positive or negative h index. Correction factors were calculated separately for each of these 6 scaling groups as an anisotropic function based on the resolution and direction in reciprocal space of each reflection. Local scale factors from this procedure ranged from 0945 to 1.00, suggesting that some systematic error remained in the data even after the 1st local scaling procedure. The positions of the iron atoms in DgFdII were determined from an anomalous difference Patterson map (Rossmann, 1961) calculated using all Friedel pairs to 1.7 A. No assumptions about the conformation of the Fe-S cluster were necessary to interpret the map, which indicated Fe-Fe distances of approximately 2.7, 2.7 and 30 A. Attempts to refine the positions of the Fe atoms against the anomalous difference data as suggested by Hendrickson et al. (1985) were unsuccessful, therefore unrefined iron atom co-ordinates were used in phasing.

DgFdII Crystal

Resolution range Crystal size (mm3) Collection method Unique reflections R(F, replicate) No. of replicates Max. decay correction No. of standard reflections Max. absorption correction

0.2 ..

m-2.5 @lxO.2xW9 Scan 3107 0044 2213 1.58 9 1.21

695

6

A

C

0.1 .0

0.01

0.02

0.03

004 005 (sin 8/X)’

006

DO7

0.08

C )9

Figure 2. Fraction of observed reflections as a function of resolution in DgFdII data set. The fraction of the total possible reflections that were retained in the final data set is shown as a function of (sin e/n)’ by open triangles. Missing reflections either were not measured or were discarded because the F2 values of replicate measurements of the reflection differed by more than 3 a( F’). The fraction of reflections for which the magnitude of the structure factor was greater than either 2 or 4 times its estimated standard deviation are indicated by open circles and filled circles, respectively. The value for ,,each shell is plotted at the average (sin e/n)’ of shell. The resolution ranges of the data collected from each crystal are also shown. Discontinuities that occur between crystals reflect the differences in crystal size and data collection techniques. (These co-ordinates differed from the final refined positions by only 62 A, uniformly in the -y direction.) The positions of the 3 iron atoms were used to calculate initial phase estimates for the reflections. Following Hendrickson’s procedure (Hendrickson et al., 1985) the generally bimodal probability distribution for the phase angle of each reflection was derived from the anomalous scattering information. The phase ambiguity was resolved by combining this phase information with that derived from knowing the positions of the iron atoms. If the phase distribution from anomalous scattering was sharply unimodal, that information alone was used. If the partial structure information allowed a clear choice of 1 of the 2 peaks in bimodal distribution, that peak was chosen. Otherwise, the phase information from the anomalous scattering and partial structure were combined. Initially, equal weight was given to phase information from the 2 sources. The additional ambiguity in the structure determination due to not knowing the absolute configuration of the

Table 1 data collection A

II

of D. gigas Ferredoxin

summary 13 2.6-2.0 0.1 x0.2x0.8 Scan 3655 0087 527 1.70 15 1.89

C 2.3- 1.7 0.1 x 0.3 x 03 stop 6228 0.090 438 1.45 15 1.57

696

C. R. Kissinger

et al.

Figure 3. Stereo view of initial electron density map from resolved-anomalous phasing. The map in the region of the Fe-S cluster is shown with the final positions for the cluster atoms and three cysteine ligands superimposed. Only electron density near the displayed atoms is shown. The map is contoured at 1.8 Q where Q is the r.m.s. deviation of the density in the map. The overall figure of merit for 4137 reflections was @53, where 38.6% were phased by “choice” with a figure of merit @76, and 61.4% were phased by “combination”, with a figure of merit of 038. Inorganic sulfur atoms Sl, S2 and S3 and the 3 cysteine Sy atoms were positioned approximately from this map and the data were rephased.

anomalous scattering atoms was resolved by calculating electron density maps using both possible arrangements of the 3 iron atoms. The electron density maps were displayed on an Evans & Sutherland PS340 graphics system using a version of the interactive molecular graphics program FRODO (Jones, 1978) supplied by P. Evans as a part of the Cambridge Crystallographic Package of computer programs (CCP4, 1979). A structural model of P. aerogenes ferredoxin was superimposed on each of the maps. The 4Fe4S cluster (cluster 1) was superimposed on the site of the cluster in DgFdII, and the P. aerogenes molecule was rotated to produce the best fit to the features of each of the electron density maps. One of the maps had electron density in the region of the cluster that was more consistent with the expected positions of the sulfur atoms and surrounding residues. Electron density for the Sy atoms of the 3 cysteine ligands and for 3 of the 4 inorganic sulfur atoms in the cluster was visible (Fig. 3). These sulfur atoms were positioned, and a new electron density map was calculated using the anomalous scattering and positional information from these atoms in addition to that of the iron atoms. This new electron density map could be interpreted clearly in the region near the cluster. The positions of the 3 cysteine residues that bind the cluster were apparent, as were several residues adjacent to them. It was clear, with the P. aerogenes ferredoxin molecule superimposed on the density, that the general chain fold near the cluster was much the same in the 2 molecules. An initial source of concern was an apparent elongation of the electron density in the direction of the b axis (coincidentally nearly normal to the plane of Fe atoms). This could not be traced to any error in the calculations and was in fact the first indication of highly anisotropic distribution of average structure factors, later attributed to anisotropic thermal motion in the crystal (discussed below). The electron density maps used throughout the model fitting procedure were calculated using data to the highest rosolution available (1.7 A). The structural detail present in these maps aided interpretation of the electron density even at the earliest stages. Lower resolution maps (e.g. 2.5 A) were no more revealing of general features of the chain folding and were far less useful for determining the conformations of individual residues. Using the superimposed P. aerogenes molecule as a guide, it was possible to fit several residues in the vicinity

of the cluster. These residues were near the 2 termini of the chain and it was readily apparent even in this early map that some of these residues formed a short 2-stranded anti-parallel /?-sheet. After 10 residues (residues 4 to 7 and 51 to 56, which except for valine 4 were modeled only as alanine residues) were fit, the data were rephased using this additional partial structure information and a new electron density map was produced. Two additional residues were fit to this new map and the data were again rephased. This iterative approach to fitting residues to the electron density was continued until most of the structure was determined. Amino acid residues were added only when there was clear density for them. Each new map invariably showed features not present in previous maps and allowed fitting of more residues. An example is shown in Fig. 4. In all, 36 electron density maps were produced. After a large portion of the structure had been fitted, the relative weight given to the phase information from the partially known structure used in the calculations of phases was reduced from I.0 to 06, which resulted in slightly more interpretable maps, presumably because the existing (imprecise) partial structure otherwise dominated the phase calculation and the resulting electron density map.

(c) Refinement Refinement of the structural model was begun when the main chain was completely traced except for the N-terminal proline residue and 12 side-chains that lacked clear density. The refinement program used was PROFFT version 2.2 (Finzel, 1987; Sheriff, 1987), which implements the stereochemically restrained least-squares refinement procedure of Hendrickson & Konnert (1980) using either conventional analytical or much faster fast Fourier transform (FFT) methods of structure factor calculation. A FFT routine was not available for space group C2, however, so the program was used in non-FFT mode. The weights and ideal values used for the stereochemical restraints are listed in Table 2. All reflections were weighted equally throughout the refinement. The weight given to the reflections during each cycle of refinement was approximately one-half of the current average AF (F,-F,), but was adjusted occasionally so that the deviations from ideal stereochemistry and differences between

Crystal Structure of D. gigas Perredoxin II

697

(b)

Figure 4. Improved electron density for valine 33 in stereo. (a) Electron density in the region of valine 33 in the first resolved-anomalous phasing map. (b) Electron density after approximately 60% of the protein atoms had been added but residues 32 and 33 were not yet in the model. In both views the final molecular model is superimposed.

observed and calculated structure factors were simultaneously minimized. For refinement, all Friedel pairs were averaged, and anomalous scattering was not considered in the calculation of model structure factors. Reflections in the resolution range co to 10 A were also excluded from the refinement because of the large contribution from disordered solvent expected for these reflections. In an effort to ensure that the radius of convergence was commensurate with errors in the initial model, refine-

Table 2 Summary

of rejinement

R-factor without overall anisotropic B correction Resolution range (A) No. of reflections No. of protein atoms No. of solvent atoms No. of variable parameters Average F,,- F, (electrons) Average B of protein atoms (8’)

results 0,157 @223 8@-l-7 4508 429 55 1941 24 16.4

Final overall anisotropic thermal factor correction (A2): -2.77 B11 cmo 812 ON 813 1261 B22 040 823 -454 B33

ment of DgFdII was begun using only data to 25 A resolution and was extended in 92 A increments after every 3 cycles of refinement until the full resolution of the data was reached, The conventional crystallographic R-factor for the starting model was 6392 for data in the resolution range 10 to 25 A (F > 2a,). The linear correlation coefficient for the same data was 6631. Because the geometry of the 3Fe-4S cluster in the molecule was of particular interest, it was desirable to refine the positions of the atoms in the cluster without the application of stereochemical restraints. This is possible if the resolution of the data is sufficient to resolve individual atoms of the cluster. The shortest interatomic distances in the cluster were expected to be the Fe-S bond lengths of approximately 2-2 to 2.3 A. As the resolution of the data extended to 1.7 A, it was feasible to refine positions of cluster atoms without restraints. At the beginning of refinement, however, the 3 Fe-P bonds were restrained to distances of 2.2 A until the resolution range was extended to 1.9 A in an effort to prevent distortions of the cluster due to insufficient resolution. No restraints were applied to the iron and inorganic sulfur atoms at any later point in the refinement, but Sy-CB bond length restraints were applied. Nine cycles of refining only positional parameters, while gradually increasing the resolution range of the data to 2.1 A, brought the R-factor to O-328 for data to that resolution. The structure was then corrected and completed by examining 2F, - Fc and AF maps using the program FRODO. A series of “delete” maps in which segments of 5 to 10 residues were removed from the structure and

698

Figure 5. Electron density shown in a resolved-anomalous

C. R. Kissinger

et al.

for side-chain of residue li6 in DgFdII in stereo. The electron density for the residue is phasing map calculated when the residue was modeled aa an alanine only.

several cycles of refinement were performed before calculation of the model electron density also proved useful in correcting misplaced portions of the model. After most of the remaining structure had been fit and the R-factor had decreased to 0305 for data from 10 to 1.7 Bi, individual temperature factors were refined for all atoms. Restraints were applied to restrict differences between temperature factors of adjacent atoms (Hendrickson & Konnert, 1980). No restraints were applied to the temperature factors of the Fe and S cluster atoms. After several cycles of refining positional parameters and individual B values for all atoms, peaks were observed in the AF map bracketing the iron and sulfur atom sites in the y direction, suggestive of highly anisotropic thermal motion. The alignment of all these peaks along the b axis suggested that all atoms in the molecule exhibited similar anisotropic mobility. but the effects appeared most strongly around the heavy atom simply because of their greater scattering power. This was consistent with the elongation of electron density in the direction of the b axis, which was observed in the 1st electron density map. A subsequent survey of the data showed more rapid fall-off in the b direction. In addition. variability of nearly 1 A in the length of the b axis observed for different crystals (data not shown) suggests that static disorder in the crystals might be responsible for the effect rather than anisotropic thermal motion, per se.

An overall anisotropic temperature factor correction for the molecule as a whole was calculated (Sheriff & Hendrickson, 1987) and was applied to the calculated structure factors in the refinement program. The application of this correction resulted in a reduction in the R-factor from 0.271 to 0.237 and improved the appearance of the AF maps considerably. The correction was recalculated every few cycles of refinement but changed little through the course of the refinement. The final values are included in Table 3. It should be emphasized that the values reported reflect a difference from the overall B, so that negative values for t,wo Bii terms are not physically unreasonable (Table 2). It became apparent at this stage of the refinement that there is an additional residue in the molecule not reported in the amino acid sequence (Bruschi, 1979). Electron

density was present beyond the Cl-terminal serine residue that had a position and shape clearly indicative of an additional amino acid residue. Because the side-chain of isoleucine Xi was well fit to electron density, but, the sidechains of the 2 existing residues beyond it, were not. it was possible that, the additional residue was present at any of the last 3 sequence positions. A “delete” map in which the side-chains of the 2 existing C-terminal residues beyond isoleucine 5Fi were removed before t,he calculation revealed side-chain electron density indicating that the previously

Table 3 S’ummary of restrained parametersfor IjgFdf I after jinal refinement cycle Restraint

IXstances (A): IIond Angle

0

N

0.019

@020 @030 0050

427 581 125

O-012

0020 0.020

58 I

0186 0187 0187 0162

0500 0.500 0.500 0.150

159 125 42 70

0041 0053

Planar I 4 Planar groups (A): Peptides

(I.016

Other Non-bonded contactst Single torsion Multiple torsion Possible H-bondsf Chiral volumes (A3)

AD

(A)

Torsion angles (“). Planar Staggered Orthonormal

2.7 193 O-2

3.0 150 20.0

58 60 1

Temperature factors (AB. AZ): Main-chain Main-chain Side-chain Side-chain

bond angle bond angle

I.75 2.67 4.37 636

24X) .wo 3@0 4+0

248 316 178 265

AD is the root-mean-square (r.m.s.) deviation from the ideal value for the parameter. 0 is the estimated standard deviation of t)he ideal value as measured in accurately determined small molecules. N is the number of restraints of this type applied during refinement. t Includes contacts between symmetry-related molecules. $ Includes hydrogen bonds between solvent atoms.

Crystal

Figure presence in which group is

Structure

of

D. gigas Ferredoxin ZZ

699

6. Electron density for side-chain of cysteine 11 in DgFdII in stereo. The electron density indicating the of an additional chemical group bonded to the Sy atom of cysteine 11 is shown. The map is part of a delete map residues 6 to 12 were removed from the model. The final molecular model with the additional methanethiol superimposed.

undetected residue was present at position 56. The sidechain density for the new residue was compatible with a valine or threonine (Fig. 5) and the side-chains of the 2 subsequent residues, which in their previous positions had been difficult to fit, now had electron density consistent with arginine and serine. Refinement of the inserted valine residue as a threonine led to a large value ( > 40 A*) for the temperature factor of the threonine Or’ atom indicating that the level of electron density was not compatible with an oxygen atom at this location. The temperature factor of the V’ atom of the valine, on the other hand, refined to a value of approximately 23 A*, in line with the temperature factors of approximately 18 A* for the Cy2 atom and 19 A* for the C? atom. Another unexpected finding in the electron density maps was additional density adjacent to the Sy atom of cysteine 11. It was clear that this residue was modified by some small chemical group. Cysteine 11 presumably is the 4th ligand of the 4-iron cluster in ferredoxin I of D. gigas, but in DgFdII it is rotated away from the cluster and is at the surface of the molecule. The electron density observed in the region of cysteine 11 is shown in Fig. 6. The additional density was centered roughly 2.0 A from the Sy atom of cysteine 11 and was the largest density peak in the AF map. It is not possible unambiguously to identify an unknown chemical group such as this crystallographically, but some indication of the atom types involved can be obtained from apparent bond lengths and by observing the temperature factor behavior when different atom types are placed in the electron density. Temperature factors of atom positions with carbon, nitrogen and oxygen scattering factors refined to values of less than 8 A*, well below the temperature factors of protein atoms in the area, suggesting that these atom types have too few electrons to flt the electron density adequately at the site. The temperature factor of a sulfur atom placed at this position refined to a value near 20 A*. The position of the additional sulfur atom was refined without any stereochemica1 restraints. Additional density adjacent to the sulfur atom was modeled as a carbon atom (methyl group), and was restrained to a distance of 1.81 A from the added sulfur atom, the approximate length of a carbon-sulfur bond. The temperature factors of the additional sulfur and carbon atoms refined to final values of 21.6 and 21.5 A*, respectively. The final temperature factor of Sy of cysteine 11 was 165 A*. These values are reasonably consistent with the expected temperature factors for adjacent atoms at the protein surface. The final refined

position of the additional sulfur atom was 229 A from the crysteine Sy atom. After the R-factor had dropped below 629, solvent molecules were added to the model whenever they were clearly discernible in the electron density maps and had a good hydrogen bond distance and geometry to an appropriate atom of the protein. Water molecules were initially assigned occupancies of 1.0 and only temperature factors were refined. In the final stage of refinement, the solvent model was re-evaluated as described below. After the structural model was largely complete and the overall R-factor had decreased to 9195, an examination of the R-factor for specific shells of data showed that the data in the 10 to 8 A resolution range had a much higher R-factor (6389) than subsequent higher-resolution shells. This suggested that the solvent continuum not included in the structural model had a relatively larger effect on these reflections and the 29 reflections in this shell were excluded from the refinement. Although the positions of all main-chain atoms were clearly visible in the electron density maps, several sidechains had poorly defined electron density even after extensive refinement, apparently because of a high degree of thermal motion, or the occurrence of multiple conformations of these side-chains. The terminal carboxyl groups of 3 glutamic acid side-chains (residues 16, 27 and 44) did not have clearly discernible electron density and could not be adequately modeled. The solvent model was re-evaluated as the final step in the refinement. There is evidence both for (Bhat, 1989; Jensen, 1990) and against (Kundrot & Richards, 1987) refinement of both occupancy and thermal solvent parameters at a resolution of 1.7 A. In this case, the occupancy was estimated on the basis of the relative height of the electron density peak at the solvent site and was fixed at this value. Only the temperature factor was subsequently refined. To assign the occupancies, all water molecules were removed from the molecular model and a AF map was calculated using data from co to 1.7 A. The highest peak in the map was considered to represent a water molecule with a full occupancy. The range of electron density from zero to the maximum peak height of approximately 69 e/A3 was divided into 10 equal intervals, and water molecules were then reassigned to peak positions and given a discrete occupancy value depending on which electron density interval the peak fell in. In general, water molecules were placed only at peaks whose heighi corresponded to an occupancy of 0.5 or above.

700

C. R. Kissinger

et al.

A few solvent sites were assigned at an occupancy of 0.4 if the electron density was well defined and the potential water site was in a good hydrogen-bonding position with an appropriate atom of the protein. After the solvent model had been completed, refinement was continued for several cycles until refinement had converged. The co-ordinates have been deposited in the Protein Data Bank, under the accession number 1FXD.

0.20

0.15

,’

. .’ ,p-..,<*,-’

.

3. Results and Discussion (a) MaEel accuracy The final refinement statistics are summarized in Tables 2 and 3. The final R-factor for 4508 reflections in the resolution range from 8.0 to 1.7 A (F > 20,) is 0157. The linear correlation coefficient for the same data is 6959. (The correlation coefficient is defined as:

For all of the data (5180 reflections) with no resolution or c cutoffs, the R-factor and linear correlation coefficient are O-192 and 6899, respectively. The average error in the atomic positions after restrained least-squares refinement estimated from a plot of the R-factor as a function of resolution (Luzzati, 1952) is shown in Figure 7. The distribution of R-factors for the DgFdII data agrees reasonably well with predicted distributions except for the lowest resolution shells, which are heavily affected by solvent not included in the model. The average co-ordinate error for the DgFdII model estimated by this method is approximately 0.15 A. The ratio of Zor/IF,l is also plotted in Figure 7. Given the relatively low values for this quantity, it is unlikely that the overall precision of the data is a limiting factor in the refinement. The estimated errors in the positions of the atoms of the Fe-S cluster are of particular interest. One estimate of the accuracy of the positions of these atoms can be obtained by performing unrestrained full-matrix least-squares refinement of these atoms alone, using the final positions of atoms in the polypeptide portion of the molecule to calculate a partial structure contribution to which is added the structure factors calculated for the cluster atoms. The standard deviations in atom positions can then be calculated from the inverse matrix of the leastsquares equations (see Cruickshank, 1965; Stout & Jensen, 1989) providing a probable lower bound on the error in these atom positions. Full-matrix leastsquares refinement of the positions of the cluster atoms was performed using the program CRYLSQ, in the XRAY76 system of crystallographic programs (Stewart et al., 1976). The estimated standard deviations in the three iron atom positions were all similar and were approximately 0020 A in the x and z directions and 6025 A in the y direction. The standard deviations in the four sulfur atom positions were approximately 6035 A in the x and z directions and O-040 d in the y direction. The

,’

\ Cl+10

, $1, _’

,I’

,’

,’

.

;r:Qi

i .

.,I’ ’ ,’

/

i

7

0

0. I

0.2

0.3

0.4

0.5

0.6

0.7

2sin(B)/X

Figure 7. R-factor as a function of resolution for DgFdII data. The R-factor for the 4267 acentric reflections was calculated in shellsof equal volumes of reciprocal space. The R-factor is plotted at the average resolution of each shell. Broken lines indicate the predicted curves calculated by Luzzati (1952) for 2 specific levels of mean co-ordinate error (in A). Also

plotted (continuousline) is the averageo/F for eachshell.

greater estimated standard deviations in the y direction are consistent with the anisotropic temperature factor behavior. Two representative views of the final 2F0- ji map are shown in Figure 8. All of the main-chain atoms are present in well-defined electron density. Only the side-chains of some external glutamic acid residues are in poorly defined density. The rootmean-square (r.m.s.) deviation of the electron density in the final AF map is 608 e/A’. The two highest peaks in the final AF map are shown in Figure 9. The highest peak (O-46 e/A3) occurs within the interior of the protein roughly 4 A from the nearest atom of the Fe-S cluster (S4). It is unlikely that this peak is due to an internal water molecule because the nearest atom, the carbonyl oxygen of residue 46, is 2.0 A away, and in an unsuitable environment for a hydrogen bond. The relatively low temperature factors of the surrounding atoms (8 to 14 A) are not indicative of disorder in this region of the molecule. Some residual electron density present in the AF map near the site of the missing fourth iron atom of the cluster (Fig. 10) might be due to the presenceof a small population of molecules in the crystal that contain the four-iron form of the cluster. The rat*io of the peak height of this position to that when an iron atom was removed from the cluster suggested that no more than one in ten molecules in the crystal would contain a four-iron cluster to result in a peak of this size. There is no indication of an alternative chain conformation around the cluster. necessary to accommodate a four-iron cluster (see below), but electron density for the lighter protein

Crystal

Structure

of D. gigas Ferredoxin

II

701

(b)

Figure 8. Final 2Fo- F, map for DgFdII in stereo. (a) A well-defined region of electron density showing residues 21 to 24. The map is contoured at 1.8Q, whereQis the r.m.s. deviation of the density in the map. (b) A lesswell-definedregion of density showingresidues56 to 58. The map is contoured at @9(I.

atoms at one-tenth occupancy would not be expected to appear significantly above the background noise level of the AF map. There are no crystal packing interactions that involve this part of the molecule, so alternative chain conformations in this region are possible without causing substantial disorder. In fact, thermal factors of atoms in cysteine 11 are not unexpectedly high (see Fig. 20 below), as would be expected if this region were disordered. (b) Overall chain folding and conformational angles A stereo plot of DgFdII is shown in Figure 11. The overall chain fold is similar to that of the three other bacterial ferredoxins of known structure. Crystal structures are now known for three-iron (this study), four-iron (Fukuyama et al., 1988), seven-iron (Stout et al., 1988; Stout, 1989), and eight-iron (Adman et al., 1973; Backes et al., 1991) ferredoxin molecules. All share the same overall folding pattern despite differences in the number and type of Fe-S clusters. A comparison of the chain-folding with that of P. aerogenesferredoxin (PaFd) is shown in Figure 12. The overall folding of the two molecules is remarkably similar. The only

regions that differ markedly between the two molecules are in the loop region comprising residues 26 to 31 in DgFdII, where there is an insertion of four residues relative to PaFd, and in the region near the second cluster of PaFd, where a disulfide bond occurs in DgFdII. A Ramachandran plot of the main-chain torsion angles is shown in Figure 13. There are no residues with highly unfavorable conformations. Alanine 10, glutamic acid 12, glycine 28 and glutamic acid 53 lie near the energy minimum at values characteristic of left-handed cc-helix. Glycine 28 participates in an unusual “Asx” type turn (discussed below) at the surface of the molecule. The other three residues are involved in binding the Fe-S cluster. The conformations that these residues assumeappear to be essential for binding the cluster and are very similar to the conformations of comparably positioned residues in P. aerogenesferredoxin. Alanine 10 and glutamic acid 12 participate in NH . S hydrogen bonds with sulfur atoms of the cluster (discussed below) that would be expected to stabilize their conformations, while glutamic acid 53 lies between two other residues that form such bonds to the cluster (see Table 4). Figure 14 shows the distribution of values of the

702

C. R. Kissinger et al.

# ++ #A +

+ + ++ l-4 +

(a)

(b)

Figure 9. Highest peaks in final DgFdII difference density map in stereo. (a) Highest peak. (b) Second highest peak. Distances to nearby water molecules are shown. Only atoms within 8 A of the center of the peaks are shown. The electron density in both views is contoured at 032 e/A’.

Figure 10. Residual density near the fourth iron site in DgFdII star. The electron density is contoured at 028 e/w3.

in stereo. The hypothetical

4th iron site is shown by a

Crystal

Structure

of D. gigas Ferredoxin ZZ

Figure 11. Stereo view of DgFdII. The main-chain and Fe-S cluster are shown by thick lines and the side-chain thin lines. Every 10th residue is labeled. Solvent molecules are not shown.

703

by

Figure 12. Comparison of DgFdII and P. aerogenes chain folding. A stereo view of the cc-carbon backbone of DgFdII lines) superimposed on that of P. aerogenes ferredoxin (thin lines) is shown. The P. aerogenes ferredoxin coordinates are those of Backes et al. (1991). The structures were superimposed by a least-squares fit of the 3 corresponding iron atoms of the common cluster in the 2 molecules. Cluster atoms and the disulfide bond in DgFdII are also depicted. (thick

first side-chain torsion angle for those residues in DgFdII with side-chains extending beyond CB. The distribution of x1 values is in accord with general surveys of protein side-chain conformations (Janin et al., 1978; Bhat et al., 1979; James & Sielecki, 1983) in that the majority of the side-chains adopt the x1 = -60” or 180” conformations, with fewer side-chains having x1 = 60”. The x1 value for the newly discovered residue valine 56 is 60”, precisely at the stereochemically ideal value. Three residues have x1 values near 120”, which is an energetically less favorable “eclipsed” conformation. These residues are glutamic acids 27 and 44 for which the terminal portions of the side-chain could not be

identified in the electron density, and arginine 57, which also exhibits very high side-chain B-values. These Cy positions are relatively poorly determined and thus the torsion angles are not well defined. A disulfide bond in DgFdII is formed between cysteines 18 and 42. The side-chain torsion angles involved in the bond are listed in Table 5. The bonded cysteine side-chains form a right-handed spiral. This conformation was found by Thornton (1981) to be typical of “local” disulfide groups between cysteine residues closer than 40 residues in the sequence. The torsion angle around the sulfur atoms differs from the values of approximately 90” right-handed disulfides typically found in

C. R. Kissinger

704

et al. ,o I.------.-..-...

-..--.

;

120

-61

-180

Figure DgFdII.

i IS0

0

14. Tjistribution of side-chain Proline residues are indicated

torsion

180

angles

in

by cross-hatched

bars.

Figure 13.

Main-chain

conformational angles in DgFdII. Cysteine residues are indicated by filled triangles. The single glycine residue is indicated by a filled circle. All other residues are indicated by open boxes. The continuous lines show “fully allowed” conformational regions for residues with CB atoms and the N-C-Cp bond angle, z (C) = 110” (Ramakrishnan & Ramachandran, 1965). Broken lines show “partially allowed” regions resulting from relaxed van der Waals’ contact constraints and 7 (C’) = 115”.

(Thornton, 1981; Richardson, 1981), which indicates that this bond might be in a somewhat strained conformation. The disulfide bond occurs in the region of the second Fe-S cluster in P. aerogelaes ferredoxin and joins the N and C-terminal halves of the molecule (see Fig. 12). Disulfide bonds are rare in intracellular proteins such as DgFdII (Fahey et al., 1977; Thornton, 1981). The reducing conditions present in the cytoplasm, including high concentrations of the reducing agent glutathione, are thought to favor free cysteine. The disulfide bond in DgFdIT is near the surface of the molecule and would appear to be accessible to intracellular reducing agents. The observed oligomerization of D. gigas ferredoxin might, however, block access to this region of the

Table 4 NH I)

A

N12 x9

SI s2 82

Nil

N14 x10 N31 N52 N54

s4 syu SYi s50 w.50

. S interactions D

A (A) 32 32 35 3.4 35 35 35 34

H

in DgFdll A (A) 2.3 2.3 2% P5 2% 2.6 2.7 25

D-H

molecule and contribute to stable disulfide bond formation. The surface location of cysteine 42 also makes possible intermolecular disulfide bonding during oligomer formation. It is apparent that a disulfide bond in this region of the molecule is not generally necessary for the stable folding of single-cluster ferredoxins. Several other single-cluster ferredoxins, including t,hose from II. thermoproteolyticus, and L)esulfovibrio afTicanus (FdI), contain no cysteines other than those directly involved in cluster binding (see Fig. I). Tf the disulfide bond is intact in viwo, it may be important for stabilizing the folding of the protein during interconversion of the three and four-iron clusters. Consistent with this, Williams (1979) has found that disulfide bonds in some other proteins appear to allow high internal mobility while maintaining the correct chain fold. Such mobility could be necessary for cluster interconversion in DgFdII. because of the significant readjustment of the polypeptide chain in the vicinity of the cluster required to accommodate a four-iron cluster (see below). Cysteine residues are also found, however, in corresponding positions in the amino acid sequence of the single-cluster ferredoxin T from Desulfrmibrio baeahtus Norway (see Fig. I), which is not known to have a three-iron cluster form, so the disulfide bonds in molecules might be entirely these ferredoxin vestigial (c) Hydrogen

A (“) 149 154 163 162 153 153 134 168

Numbering of inorganic sulfur atoms and cysteine Sy atoms is as in Fig. 17. All nitrogen atoms are main-chain amide nitrogens and are identified by residue number. Hydrogen atom positions were calculated using the Program X-PLOR. D. donor: A, acceptor.

bonding

A representation hydrogen bonds

and secondary

of the main-chain between them

structure atoms and the is shown in

Table 5 Conformational

Residue (‘ys18 (‘~~42

angles (“) of the disul$de DgFdll

bond in

x1

x2

x3

- 153 54

55 81

-57

Table 6 Hydrogen bonded atoms by distance criteria A.

Hydrogen

bonds

involving

Side-chain

N

N...O

S 580XT

28

0 P V 0 A N

main-chain

Res. no. 0

920 360 560 840 540 50

E A A C

2.9 2.8 30 33 %9 32

31 2.6 2.9

120 130 130 140

N

250

E 0 V D 70D2

230 680 210

2.9 0 0 L

960 950 400

D

410

V 430 C 420 v 430 E 440 E 450 A 460 I 470 A 460 c 500 0 730 E 30 0 1290 P 10 B.

Hydrogen

Res. no. 68 70 72 72 73 84 85 90 92

bonds

O...N v M P D I v

33N 24N 19N 20N 55N 4N

involving

0 30 2.7 32 30 3.1 3.3

solvent

1N

29

This Table contains interatomic distance § (f-~,f+Y,-~).

and

Side-chain

D 200D2

S 580XT N 35ND2 P

N

1 s 58N 2 3 V 56N 4 5 C 8N 8 12 v 15N 13 I 17N 13 E 16N 14 c 18N 33 15 32 16 3.1 17 30 18 V 21N 18 F 22N 3.2 19 30 20 30 21 I 34N 31 22 2.9 23 V 32N 2.7 24 2.8 25 G 28N 26 3.0 28 29 30 N 25N 25 32 E 23N 30 33 28 34 36 I 2N 39 4oV43N 3.1 41 E 44N 32 42 A 46N 3.1 43 A 46N 43 I 47N 31 44 D 48N 45 S 49N 32 46 3 49N 2.9 46 C 50N 30 47 c 5oN 31 48 2.8 49 3.3 49 33 50 E 53N 3.0 50 32 53 54 N 5N 3.1 55 30 56 E 3N 30 57 28 58

0 720 0 720 C 180 C 180 V 320 0 700 K 300 E 230E2t N 250Dl N 250Dl

atoms

15. Hydrogen

0

Side-chain

2.8 3.0 K 30NZ

3.0

3.2 3.3 3.1 32 3.0

protein

30 31

Res. 110.

bonds

0

94 95 96 97 97 107 116 121 127 128 129 131 131 134 135 136 136

involving

N

C 42N D 41N

R 57N

solvent

0

2.8 3.1

protein

atoms

(cont.)

Side-chain

D 200Dl

3.3

D 8 D D R K D

70Dl 380G 390D2 370D2 57NHl 30NZ 60Dl

2.9 28 3.2 32 31 2.7 26

R R D D E E

57NH2 57NHl 200D2 200Dl 450E2t 45032

3.1 3.0 2.9 32 2.6 2.6

32 31

30

2.8 C. hydrogen

25 E 230Elt

bonds

sorted

by side-chain

32

30 no. 2.8 N250Dl 2.9

32

2.8 31 31 2.9 32 3.0 31 2.8 33 30 33

S 490G

32

2.7

Side-chain

3 6 D

main-chain

Side-chain

6OD1

0

1280

2.6

7 D D 12

70D2 70Dl

D 0

39N 970

2.9 2.9

20 D D D D 23 E E 25 N N N 26

200D2 200Dl 200D2 200Dl 230Elt 230E2t 250Dl 250Dl 250Dl

0 0 0 0 M E G D K

720 940 1340 1350 240 26N 28N 29N 300

2.8 33 29 32 32 31 26 2.9 3.2

29

2.9 30

atoms

30 K K 35 N 37 D 38 S 39 D

30NZ 30NZ 35ND2 370D2 380G 390D2

V 0 0 0 0 0

40 1270 900 1160 970 1070

30 >7 31 32 28 32

45 E 450E2t E 45032 48

0 0

1360 1360

2.6 2.6

49 S 57 R R R 58 S

E 0 0 0 P

41

2.8

and

490G 57NHl 57NH2 57NHl 580XT

450 1210 1310 1310 1N

2.7 31 31 30 2.8

E D D D D

side-chain

30E2 60D2 60Dl 60Dl 70Dl

3 580G 1) 60Dlt 1) 60Dlt I) 6OD2t S 380G

2.8 31 26 31 2.9

E 12OE2 E 120E2 E 120El

D 390Dl$ 1) 390D2$ I> 390Dl$

2.6 3.1 30

E 230El

E 230Elt

31

N 25ND2

ID 29OD2

3.2

E E D D D D

26032 260El 290D2 290D2 290Dl 290Dl

R R N D D

57NEg 57NE§ 25ND2 290Dlt 290D2t -D 290Dlt

32 32 32 32 32 26

S D D D D D E

380G 390D2 390Dl 390Dl 4lOD2 410Dl 45032

D E E E D D E

70Dl 120E2$ 120Elf 120E2$ 480Dlt 480Dlt 450E2t

2.9 31 30 2.6 32 24 32

D 480Dl D 480Dl

D 410D2t D 410Dlt

32 2.4

R 57NE R 57NE

E 260E26 E 26OElg

32 32

S 580G

E

2.8

listings of all potential hydrogen bond interactions involving protein atoms in DgFdII of less than 33 A. Atoms in symmetry-related molecules are indicated by t (--s,y,-z),

30E2

as indicated 1 (f+x,$+

by an y,z) or

C. R. Kissinger

706

et al.

Figure 15. Main-chain hydrogen bonds in DgFdII in stereo. Continuous lines represent representhydrogen bondsdeterminedby criteria of Kabsch $ Sander (1983).

main-chain

atoms

and

broken

lines

Figure 15. A list of all potential hydrogen bond interactions in DgFdII involving both main-chain and side-chain atoms as indicated simply by an interatomic distance of less than 3.3 A is given in Table 6, while Table 7 gives the major secondary structural elements found in DgFdII. The occurrence of a-helix B in DgFdII is as predicted by Fukuyama et al. (1988) for single-cluster ferredoxins. One notable structural feature in DgFdII is the presence of several Asx-type reverse turns (Rees et al., 1983; Baker & Hubbard, 1984), in which there is not only the hydrogen bonding between the mainchain carbonyl group of residue n and the amide group of residue n + 4 that defines a classical reverse turn but also a hydrogen bond between a carbonyl oxygen atom on the side-chain of residue n and the amide group of residue n + 3 (see Tables 6 and 7). Two Asx turns in DgFdII are shown in Figure 16. The turn containing residues 25 to 29 (Fig. 16(b)) is somewhat unusual in that it involves no main-chain hydrogen bonding, but the side-chain carbonyl oxygen atom of asparagine 25 is clearly hydrogen bonded to the main-chain amide groups of both

Table 7 Secondary Chain segment Prol-V&l4 AsnS-Cys8 Ala13-Ile17 Cys18-Va121 Phe22-Met24 Asn25-Asp29 Ala31-Va133 Asn355Ser38 Asp4-Ser49 Cys50-Glu53 Ile55-Ser58

structural

elements

Secondary

in DgFdII

structure

Anti-perallel B-sheet A Type I/Asx turn (0, N,, 0: a-Helix A Type I turn (18 O1s.. N,,) Anti-parallel p-sheet B Asx turn (Od,; N,,, 0:s N,,) Anti-parallel b-sheet B Type I/Asx turn (O,, Nss, O”,; a-Helix B Type I/Asx turn (O,, Ns3, S&, Anti-parallel /?-sheet A

N,)

NJ,) Nsz)

glycine 28 and aspartic acid 29 with very good hydrogen bond geometry in both cases. A carbonyl oxygen atom with two hydrogen donors is a very energetically favorable bonding arrangement (Baker & Hubbard, 1984) and this turn is likely to be quite stable, despite the lack of main-chain hydrogen bond interactions. The occurrence of several Asx-type turns in DgFdII is intriguing. Only one of the five turns in the molecule is not of this type. Such turns occur frequently in proteins (Baker & Hubbard, 1984), but the presence of four such turns in a small protein such as DgFdII might be indicative of a special role for them in the molecule. The additional stability afforded by such turns could be important in limiting dynamic motion in the molecule, leading to loss of the Fe-S cluster. It is also possible that such turns are particularly common in small pro. teins such as DgFdII. Small proteins with a high surface to volume ratio have less “interior” and are held together less strongly by hydrophobic interactions and thus might require additional hydrogen bond interactions for folded stability. The turn at residues 25 to 29 contains three acidic groups and forms a rather noticable protrusion from the surface of the molecule. Of the 19 charged residues, only two are basic. The negative charge appears to be rather uniformly distributed over the surface of the molecule. The protruding turn is adjacent to the iron-sulfur center and could be important in specific interactions with electron transfer partners, such as cytochrome c3 although one must keep in mind that the reactive speciesare dimers or tetramers. Such an interaction has been modeled for the ferredoxin-cytochrome es pair from Desulfovibrio vulgaris Norway (Cambillau et al.. 1988). (d) Three-iron

cluster and its binding

to protein

Figure 17 portrays the three-iron complex (the cluster plus the 3 cysteine Sy ligands) including

Crystal

Structure

of D. gigas Ferredoxin

II

707

(b)

Figure 16. Asx-type turns in DgFdII in stereo. Hydrogen bonds are indicated by broken lines (a) Turn involving residues 5 to 8. (b) Turn involving residues 25 to 29.

values of individual bond lengths and angles. As shown in Table 8, average values of bond lengths and angles are very close to those in the four-iron complexes of Chromatium virwsum high-potential iron protein and P. aerogenes ferredoxin. None of the average values for corresponding bonds and angles in the three structures differs significantly. The standard deviations in the positions of the atoms of the cluster can be estimated from the variation in bond lengths involving those atoms. If the standard deviation in position of a sulfur atom is assumed to be twice that of an iron atom, then, through propagation of error, the variance in position of an iron atom is roughly equal to one-fifth, and the variance of a sulfur atom is roughly equal to four-fifths, of the variance of the Fe-S bond lengths. The positional standard deviations estimated from this method are roughly 902 A for the iron atoms and 004 A for the sulfur atoms. These values are in remarkably good agreement with those calculated from the least squares matrix from refinement of the

cluster atoms (see section (a), above). There are no significant differences, within the precision of this study, between the bond lengths and angles involving the single trivalent sulfur atom in the cluster and those involving the divalent sulfur atoms. The geometry found for the 3Fe-4S cluster in DgFdII is consistent with the body of spectroscopic evidence for this and other ferredoxin molecules containing three-iron clusters. The three-iron cluster in Azotobacter vinelandii ferredoxin I, as redetermined (Stout et al., 1988; Stout, 1988, 1989; Merritt, Stout & Jensen, unpublished results), has geometry that is essentially the same as that of the cluster reported here. It seems likely that most, if not all, three-iron clusters in proteins share this configuration under normal conditions, although a linear form of three-iron cluster is reported to form in aconitase at pH > 9 (Kennedy et al., 1984). No model compounds containing this type of 3Fe-4s cluster have yet been synthesized, although

708

C. R. Kissinger

Figure 17. Iron-sulfur complex in DgFdII. The numbering of iron and inorganic sulfur atoms corresponds to that of equivalent atoms of P. aerogenes ferredoxin complex I (Backes et al., 1991). The numbering of SY atoms refers to the sequence position of t,he cysteine residue. Bond distances are in 8.

anion contains the “rose-window” Nd’edb 3Fe-4S units within it, with geometry not unlike that found here (You et al., 1990). It is possible that the surrounding protein environment is essential for maintaining the structure of a single 3Fe-4S cluster. Holm and colleagues have recently reported the synthesis of a “spin-isolated” 3Fe-4S cluster fragment that. although not structurally characterized because suitable crystals were lacking, has been shown to have spectroscopic properties remarkably similar to those of DgFdII (Weigel et aZ., 1990). Although part of an 4Fe-4S core, the spin-isolated fragment is effected by a large tri-thiol ligand that forces trigonal geometry. It appears that imposition of trigonal symmetry is sufficient to form the spincoupled ground state characteristic of the cubaneminus-a-corner 3Fe-4S centers, leading these authors to conclude that the ground state is an inherent property of the center “not conditioned by elements of the protein structure”. The Fe-S cluster is bound to the polypeptide chain through cysteine residues 8. 14 and 50. As in A. vinebndii ferredoxin I, cysteine 11 is dissociated from the cluster. It is rotated away from the cluster and is modified by the addition of a methanethiol group (as described under Experimental Methods, section (c), above). The three divalent inorganic sulfur atoms of the cluster and two of the cysteine Sy atoms are involved in NH . . S hydrogen bonds with mainchain amide nitrogen atoms of the protein (Table 4, Fig. 18), as has been found in 4Fe-4S-containing ferredoxins and other Fe-S proteins (Carter et al., 1974; Adman et al., 1975; Fukuyama et aZ., 1988). The single trivalent sulfur in the cluster is not involved in hydrogen-bonding and lies in a non-

et al.

polar environment. This is also true of the c’orresponding sulfur atom of the four-iron cluster ot P. aerogenes ferredoxin. The covalent environments of the three Fe atoms in the cluster are equivalent. This is consistent with the results of Mossbaurr spectroscopy (Huynh et al., 1980), which indicated three equivalent Fe atoms in tetrahedral environments in the oxidized form of t,he cluster. Those studies also suggest. however, that upon reduction of the cluster, the added electron is localized on only two of the iron atoms. It is possible, as suggested by Beinert & Thomson (1983), that a covalent rearrangement accompanies reduction that leads to spectroscopically non-equivalent iron atoms. Such a readjustment would also be consistent, with the large changes observed in the resonanc>e Raman spectrum upon reduction of the clust,er (Johnson I+ al., 1981). in fanvironmerit Differences the protein surrounding the cluster also might lead to localization of the electron on only two of the iron atoms. There is asymmetry in the arrangement of NH S bonds around the cluster (see Fig. 18). The Sy at’orns of cysteines 8 and 50 each have two such bonds while that of cvsteine 14 has none. The influence of these bonds might play a role in determining the electronic configuration of the reduced cluster. Another possible factor affecting electronic structure is the conformation of the cysteine ligands.

Comparison

of bond

Table 8 lengths and angles in three-iron

and four-iron

clusters

DgPdII A. Bond lengths Fe

Fe

s...s

Fe-S

FP t-7

B. Bond an&s Fe-S-Fe

S-Fe-S

&Fe-S

HiPIP

PaPd

2.72 2.68..2.78 @04 3.55 :949-3%4 096 4.26 2.10-2.39 om 220 237-222 092

I69 255-2.79 WOH 35.5 3.44~-384 0.07 2.25 2.16.-2.37 0.0.5 %30 2.24-2-38 0@5

(A)

Mean Range r.m.s Mean Range r.m.s. Mean Range r.m.s. Mean Range r.m.s.

drv.

dev.

dev.

dev.

275 2.70-2.79 004 3.63 356--3.7 1 0.05 2.28 2.22-2.33 0.04 2.26 2%?~3~29 003

(deg.) Mean Range r.m.8. dev. Mean Range r.m.s. dev.

Mean Kange r.m.s.

de\--.

74 71-77 1.7 105 101-100 2.2 114 lot-121 46

74 72-~76 1.3 104 101-109 2.4 115 107~120 4.9

74 70-7X 2.0 104 IO& 109 2.3 114 10% 124 6.5

Listed are the equivalent bond lengths and angles in the Fe&S: complex of Il. gigas ferredoxin (DgFdII) and the Fe,S& complexes of Chromatium vinosum high-potential iron protein (HiPIP) and P. uewgenes ferredoxin (PaFd). Values for HiPIP are from Carter (1977). Values for PaFd are for both complexes in the molecule and are from Backes it al. (1991).

Crystal Structure

of D. gigas Ferredoxin

II

709

(b)

Figure 18. Pu’itrogen-sulfurhydrogen bondsin DgFdII in stereo. Hydrogen bonds are indicated by broken lines.C” and Fe atoms are labeled.

Backes et al. (1991) have shown that it is likely that the frequency and strength of Resonance Raman vibrations associated with the Fe-S bonds are sensitive to the relative orientation of the Fe-S-Cfi-C” dihedral angles in 4Fe-4S centers in ferredoxins and high potential iron proteins. In the present molecule, two of the cysteine residues have approximately the same conformation (e.g. 8 and 50 have torsion angles of 68” and 88”) whereas the third is quite different (260”). Chakrabarti (1989) has pointed out that these angles tend to fall in clusters in metalloproteins. The present pattern is not outside these observations, but does suggest which two of the three iron atoms are more likely to be equivalent. The corresponding angles in P. aerogenes ferredoxin are 62”, 61” and 86”, 84” for residues 8, 36 and 46, 18, while the angle corresponding to residue 14 is 244” and 252”, and the remaining residues are 302” and 298” (Backes et al., 1991). The theoretical results of Borshch & Chibotaru (1989), however, indicate that electron delocaliza-

tion over two of the iron atoms can occur via vibronic interactions in the absence of significant structural non-equivalence of the three iron atoms. In the light of the spectroscopic results, the crystal structure of the reduced form of DgFdII, if it can be obtained, should prove instructive. Despite the similarity in geometry between the 3Fe-4S clusters from D. gigas and A. vinelandii ferredoxins, there is a large difference in reduction potential reported for the two clusters: - 130 mV for DgFdII (Cammack et al., 1977) and -420 mV for A. vinelandii ferredoxin I (Sweeney et al., 1975). Carter (1977) has noted a correlation between the number of NH . . . S hydrogen bond interactions and reduction potential in the 4Fe-4S ferredoxins and has estimated an 80 mV increase in reduction potential for each hydrogen bond. Stout (1989) reports four such interactions for the 3Fe-4S cluster in A. vinelandii ferredoxin I, while the cluster in DgFdII has eight. This leads to the prediction of a redox potential for A. vinelandii ferredoxin that is 320 mV more negative than DgFdII, which agrees

710

C. R. Kissinger et al.

Figure 19, Comparison of chain conformation around cysteine 11 for DgFdII and P. aerqenes ferredoxin in stereo. The DgFdII polypeptide chain is shown by thick lines and the P. aerogenesferredoxin chain is shown by thin lines. The structures were aligned as in Fig. 12.

quite well with the observed difference of 290 mV. On the other hand, Backes et al. (1991) have tabu-

similar in three of the cited factors are also important.

lated

Interconversion between the three-iron and fouriron clusters would require a significant readjustment of the cysteine side-chain, and some adjustment of the main chain. Cysteine 11 is rotated so

redox

potentials ranging from -280 mV -645 mV stearothermophilus) to (A. vinelandii) for a number of four-iron ferredoxin molecules, and the hydrogen bonding is known to be ( BaCilluS

Temperature

cases.

Clearly

other

factor

Figure 20. Distribution of individual temperature factors in DgFdII. The histogram atoms that have temperature factors falling in a given range.

depicts the number of protein

Crystal

Structure

of D. gigas Ferredoxin

that the Cfl atom is in van der Waals’ contact with the cluster and the Sy atom is rotated away from the cluster. A comparison of the chain conformations around the three-iron cluster of DgFdII and the analogous four-iron cluster of P. aerogenes ferredoxin is shown in Figure 19. The largest differences in position are found only for cysteine 11 and immediately adjacent residues. Thus the conversion between cluster forms would probably involve only a local adjustment of the chain. Residues 5 through 8 are involved in a hydrogen-bonded Asx-type turn, while residues 13 through 17 are in an a-helix, both of which would probably be resistant to large conformations1 changes. This also suggests only local conformational changes. The chemical modification of cysteine 11 has obvious implications for control of cluster type in the molecule. It remains to be determined whether this modification occurs in vivo or is an artifact produced during purification of crystallization. The presence in a bacterial protein of a cysteine residue modified by the addition of a methanethiol group is not unprecedented. This modification has been found in a streptococcal proteinase (Lo et al., 1984), where it appears to play a role in secretion of that protein. We note that methanethiol has been found as a product of D. gigas metabolism (Hatchikian et al., 1976), suggesting that this compound or another sulfur metabolite is the modifying agent. Chemical characterization of the modification is underway (J. LeGall, personal communication).

II

711

Conversion from a four-iron to three-iron cluster can occur in D. gigas ferredoxin in vitro under conditions that are unlikely to involve modification of a cysteine residue (Moura et al., 1982). It is possible, however, that covalent modification of cysteine 11 in DgFdII is required for maintenance of the three-iron cluster under physiological conditions. This suggests a possible enzymatic mechanism for the interconversion of DgFdI and DgFdII in D. gigas cell extracts observed by Moura et al. (1984). (e) Temperature factors The distribution of individual temperature factors of the protein atoms is shown in Figure 20. Although the average temperature factor of the protein atoms, 16.4 A2, does not agree well with the overall temperature factor of 12 A2 calculated from the Wilson plot, the peak in Figure 20, at 11 to 12 A2, and the median temperature factor 136 A2 do correlate well with the temperature factor from the Wilson plot. The average temperature factors for the mainchain and side-chain atoms of each residue are shown in Figure 21. As expected, the temperature factors are highly correlated with the relative exposure of a residue to solvent. The highest temperature factors are found on glutamic acid and arginine side-chains, which protrude into the solvent. In general, the highest main-chain temperature factors

60

Figure 21. Temperature factors of main-chain and side-chain atoms in DgFdII. Main-chain (continuous line) and sidechain (broken line) temperature factors are shown for each residue. Asterisks indicate incompletely modeled side-chains. Cysteine residues are indicated by triangles near the bottom margin of the plot. Also depicted is the fraction of the surface area of each residue that is exposed to solvent (uncorrected for lattice contacts) calculated by the method of Connolly (1983).

712

C. R. Kissinger

et al.

(b)

Figure 22. Water structure around DgFdII. Water molecules are indicated by dots. (a) Stereo view of’ DgFdlI with the all water molecules shown. (b) Stereo view of the 8 tightly bound water molecules (occupancy 2 ti8 and temperat,ure factor I 25 AZ. Potential hydrogen bond interactions with protein atoms and other tightly bound water molecules are shown.

are found at the chain termini and in the highly exposed loop consisting of residues 23 to 30. The lowest temperature factors belong to residues which are mostly or entirely buried in the protein interior. Temperature factors for the cluster atoms range from 9.7 to 12.6 A2. (f) Solvent structure The estimated solvent content of the crystals is roughly 33% by volume. Assuming the volume occupied by a water molecule to be approximately 30 A3, this corresponds to approximately 125 fully occupied solvent molecules per asymmetric unit. There are 56 solvent positions included in the final molecular model. Considering partial occupancies, 33 of the 125 solvent atoms are accounted for. The small fraction of solvent atoms observed crystallographically reflects the disordered nature of the most of the water in the crystal. Nevertheless, the fraction of the theoretically present solvent identified in this study is somewhat higher than for some

other protein structures determined at similar resolution (e.g. Finzel et al., 1985; Karplus & Schulz. 1987; Read & James, 1988), despite the conservative approach taken in incorporating solvent molecules into the model. This is probably due to the relatively small size of DgFdII and the low proportion of solvent in the crystals, so that the solvent, channels are relatively small and a higher proportion of the solvent molecules interacts directly with the protein. Eight of the water molecules havr occupancies above 0.8 and temperature factors below 25 A2 and thus appear to be tightly bound to the protein molecule. These are shown in Figure 22. The majority of these water molecules are found at intermolecular contact regions formed by the packing of the protein molecules in the crystal. Two. however, are found in a groove in the protein surface between the C-terminal end of u-helix B and the turn involving residues 50 to 53 and might be integral to the protein structure. One solvent molecule appears to be involved in a hydrogen bond interaction with the exposed Sy atom of cysteine 42

Crystal

Structure

of D. gigas Ferredoxin II

713

Figure 23. Crystal packing of DgFdII. Two stereo views of the unit cell are shown. Water molecules are indicated crosses. The a axis is horizontal and the b axis is vertical

by

along with hydrogen bonds to an oxygen and a nitrogen atom of the protein. The S . . . 0 distance is 3.44 A, which is marginally indicative of hydrogen bonding.

Crystal packing contacts are relatively sparse in the direction of the b axis, which is consistent with apparent mobility of the atoms in the b direction in the crystal structure and the variability in length of the b axis.

(g) Crystal packing

This work was supported in part by NH grants GM13366 (L.H.J.), and GM31770 (E.T.,4.). We are grateful to Jean LeGall for initiating this project with us.

Two stereo views of the crystal packing of DgFdII are shown in Figure 23. Although DgFdII is isolated as a tetramer, it crystallized as a monomer in the asymmetric unit. There are four molecules in the unit cell, but it is apparent from an examination of the crystal packing interactions that their arrangement is unlikely to be relevant to tetramer formation. There are no large contact areas between molecules. The majority of crystal contacts are intermolecular hydrogen bonds formed around the 2-fold symmetry axes of the unit cell. There are no extensive hydrophobic interactions.

References Adman, E. T., Sieker, L. C. & Jensen, L. H. (1973). J. Biol. Chem. 248, 3987-3996. Adman, E. T., Watenpaugh, K. D. & Jensen, L. H. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 4854-4858 Backes, G.. Mino, Y., Loehr, T., Meyer, T.: Cusanovich, M. A., Sweeney, w. v., Adman, E. T. & Sanders-Loehr, J. (1991). J. Amer. Chem. Sot. 113. 2055-2064.

714

C. R. Kissinger

Baker,

E. N. & Hubbard, R. E. (1984). Progr. Biophys. Biol. 44, 97-179. Beinert, H. (1990). FASEB J. 4, 2483-2491. Beinert, H. & Thomson, A. J. (1983). Arch. Biochem. Biophys. 222, 333-361. Bhat, T. N. (1989). Acta Crystallogr. sect. A, 45, 145-146. Bhat, T. N., Sasisekharan, V. & Vijayan, M. (1979). Znt. J. Peptide Protein Res. 13, 170-184. Borshch, S. A. & Chibotaru, L. G. (1989). Chem. Phys. 135, 375-380. Bruschi, M. (1979). Biochem. Biophys. Res. Commun. 91, 623-628. Bruschi, M. & Guerlesquin, F. (1988). FEMS Microbial. Rev. 54, 155-176. Bruschi, M. & LeGall, J. (1976). In Zron and Copper K. T., Mower, M. F. & Proteins (Yasunobu, Hayaishi, O., eds), p. 57, Plenum, New York. Bruschi, M., Hatchikian, E. C., LeGall, J., Moura, J. J. G. & Xavier, A. V. (1976). B&him. Biophys. A&, 449, 275-284. Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. & Bruschi, M. (1988). Proteins: Struct. Fun&. Genet. 4, 63-70. Cammack, R., Rao, K. K., Hall, D. O., Moura, J. J. G., Xavier, A. V., Bruschi, M., LeGall, J., Deville, A. & Gayda, J. P. (1977). B&him. Biophys. Acta, 490, 31 l-321. Carter. C. W., Jr (1977). J. Biol. Chem. 252, 7802-7811. Carter. C. W., Jr, Kraut, J., Freer, S. T. & Alden, R. A. (1974). J. Biol. Chem. 249, 6339-6346. CCP4 (1979). The S.E.R.C. (U.K.) Collaborative Computing Project No. 4. A suite of programs for protein crystallography, distributed from Daresbury Laboratory Warrin ton, WA4 4AD, U.K. Chakrabarti, P. (1989). 5 tochemistry, 28, 6081-6085. Connolly, M. L. (1983). Science, 22i, 709-713. Cruickshank, D. W. J. (1965). In Computing Methods in Crystallography (Rollett, J. S., ed.), pp. 112-l 16, Pergamon Press, Oxford. Eck, R. V. & Dayhoff, M. 0. (1966). Science, 152. 363-366. Fahey, R. C., Hunt, J. S. & Windham, G. C. (1977). J. Mol. Evol. 10, 155-160. Finzel, B. C. (1987). J. Appl. Crystallogr. 20, 53-55. Finzel, B. C., Weber, P. C., Hardman, K. D. & Salemme. F. R. (1985). J. Mol. Biol. 186, 627-643. Fukuyamma, K., Nagahara, Y., Tsukihara, T., Katsube, Y., Hase, T. & Matsubara, H. (1988). J. Mol. Biol. 199, 183-193. George, D. G., Hunt, L. T., Yeh, L. S. L. & Baker, W. C. (1985). J. Mol. Evol. 22, 20-31. Hanson, J. C., Watenpaugh, K. D.; Sieker, L. C. & Jensen. L. H. (1979). Acta Crystallogr. sect. A, 35, 616-621. Hatchikian, E. C., Chaigneau, M. & LeGall, ,J. (1976). In Mol.

Microbial

Production

and

Utilization

of

Gases

(Schegel, H. G., Gottschalk, G. & Pfennig, N.. eds), pp. 109-l 18, Goltze, Gottingen. Hendrickson, W. A. & Konnert, J. H. (1980). In Biomolecular Evolution

Press, Hendrickson, Methods

Huynh, B. LeGall, Chem.

Structure,

Function,

Conformation

and

(Srinivasin, R., ed.), pp. 43-57, Pergamon Oxford. W. A., Smith, J. L. & Sheriff, S. (1985). Enzymol. 115, 41-54. H., Moura, J. J. G. Moura, I., Kent, T. A.. J., Xavier, A. V. & Munck, E. (1980). J. Biol. 255,

3242-3244.

James, M. N. G. & Sielecki, 163, 299-361.

A. R. (1983). J. Mol.

Biol.

et al Janin,

J., Wodak, S., Levitt, M. & Maigret,, B. (1978). Biol. 125, 357-386. H. (1990). Acta Crystallogr. sect. B, 46, 650-654. Johnson, M. K., Hare. J. W. & Spiro, T. G. (1981).

J. Mol. Jensen, I,.

J. Biol.

Chem.

256,

9806-9808.

Jones, T. A. (1978). J. Appl. Crystallogr. 11, 268.-272. Kabsch, W. & Sander, C. (1983). Biopolymers 22, 2577-2637. Karplus, P. A. & Schulz, G. E. (1987). J. Mol. Biol. 195, 701-729. Kennedy, M. C.. Kent, T. A., Emptage, M., Merkle. H.. Beinert, H. & Munck, E. (1984). J. Biol. Chem. 259. 14463-14471. Kent, T. A., Dreyer, J. L., Kennedy, M. (‘., Huynh, B. H., Emptage, M. H., Beinert, H. & Munck, E. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 10961100. Kissinger, C. R. & Sieker, L. C. (1987). Amer. Crystallogr. Assoc. Annu. Meeting Abstr., Austin, p. 16. Kissinger, C. R., Adman, E. T.. Sieker, L. (‘. & Jensen. L. H. (1988). J. Amer. Chem. Sot. 110. 872lb8723. Kissinger. C. R., Adman, E. T., Sieker, I,. (“. , Jensen. L. H. & LeGall, J. (1989). FEHS Letters, 244. 447-450. Kundrot. C. E. & Richards, F. M. (1987). dcta (‘rystalloyr. sect. B, 43, 544-547.

Lo, S.-S., Fraser, B. A. & Liu, T.-Y. (1984). J. Biol. (‘hem.. 259, 11041-11045. Luzzati, P. V. (1952). Acta Crystallogr. 5, 802.-810. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Matthews, B. W. & Czerwinski, E. W. (1975). Acta Crystallogr. sect. A, 31, 480-487. Moura, J. J. G., Xavier, A. J., Hatchikian. E. (‘. Nr LeGall, J. (1978). FEBS Letters, 89, 177- 179. Moura, J. J. G., Moura, I., Kent. T. A., Lipscomb, .J. 0.. Huynh, B. H., LeGall, J., Xavier, A. V & Munck. E. (1982). J. Biol. Chem. 257, 6259-6267. Moura, J. J. G., LeGall, J. & Xavier, A. V. (1984). Eur. J. Biochewh. 141, 319-322. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968). Acta Crystallogr. sect. A, 24, 351..-359. Ramakrishnan, C. & Ramachandran. G. N, (1965). Biophys.

J. 5, 909-933.

Read, R. J. & James, M. N. G. (1988). J. Mol. Biol. 200. 523-551. Rees, D. C., Lewis, M. & Lipscomb, W. N. (1983). J. Mol. Biol. 168, 367-387. Richardson, J. S. (1981). Advan. Protein (Ihenc. 34. 167-339. Rossman, M. G. (1961). Acta CrystaElogr. 14, 383-388. Sheriff, S. (1987). J. Appl. Crystalkgr. 20, 55-57. Sheriff, S. & Hendrickson, W. A. (1987). dcta Crystallogr. sect. A, 43, 118-I 21. Sieker, 1,. (1.. Adman, E. T., Jensen, L. H. $ LeGall. .I. (1984). J. Mol. Biol. 179, 151-155. Smith, J. t Hendrickson, W. A. (1982). A suite of Resolved Anomalous Phasing Programs. In Computational Crystallography (Sayre. I)., rd. ) , 1). 209, Oxford Cniv. Press (Clarendon), London & New York. Stewart, J. M., Machin, P. A,, Dickenson, C. W., Amman: H. L., Heck, H. & Flack, H. (1976). The XRA Y7fi System, Tech. Report TR-446, Computer Science Center, University of Maryland, College Park. MD. Stout, C. D. (1988). J. Biol. Chem. 263, 9256-9260. Stout, C. D. (1989). J. Mol. Biol. 2OS, 545-555. Stout, 0. H. C Jensen, I,. H. (1989). X-Ray 8tructurr Determination, John Wiley, New York.

Crystal Structure

of D. gigas Ferredoxin II

Stout, G. H., Turley, S., Sieker, L. C. & Jensen, L. H. (1988). Proc. Nat. Ad. Sci., U.S.A. 85, 1020-1022. Sweeney, W. V., Rabinowitz, J. C. & Yoch, D. C. (1975). J. Biol. Chem. 259, 7842-7847. Thomson, A. ,J., Robinson, A. E., Johnson, M. K., Cammack, R., Rao, K. K. & Hall, D. 0. (1981).

Biochim. Biophys. AC.%,637, 423-432. J. M. (1981). J. Mol. Biol. 151, 261-287.

715

Weigel, J. A., Srivastava, K. K. P., Day, E. I’., Munck, E. & Holm, R. H. (1990). J. Amer. Chem. Sot. 112, 80158023. Williams, R. J. P. (1979). Bio2. Rev. 54, 389-437. Wilson, A. J. C. (1942). Nature (London), 150, 152. You, J.-F., Snyder, B. S., Papaefthmiou, G. C. & Helm, R. H. (1990). J. Amer. ChemSot. 112, 1067-1076.

Thornton,

Edited by R. Huber