The Structure of a Bacterial Ferredoxin*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 11, Issue of June 10, PP. 3987-3996, 1973

Printed in U.S.A.

The Structure of a Bacterial Ferredoxin*

(Received for publication. November 22, 1972)

ELINOR T. ADMAN, LARRY C. SIEKER, AND LYLE H. JENSEN

From the Department of Biological Structure, University of Washington, School of Medicine, Seattle, Washington 98195

SUMMARY

The structure of the bacterial ferredoxin from Peptococcus aerogenes (54 amino acids) has been determined at 2.8 A resolution. The molecule is a prolate ellipsoid with approximate dimension of 22 x 27 A. The iron and sulfur atoms are in two complexes 12 A apart with 4 iron, 4 inorganic sulfur, and 4 cysteine sulfur atoms in each. Cys 8, 11, 14, and 45 are coordinated to iron atoms in one complex and Cys 18, 35, 38, and 41 are coordinated to iron in the other. The two tyrosine groups present in the molecule are oriented in a similar way with respect to the complexes: Tyr 2 is parallel to and apparently in contact with one face of one complex and Tyr 28 is oriented similarly with respect to the other. Both tyrosine rings have an edge exposed to the solvent. The structure was solved with three isomorphous derivatives, at first used independently but later combined, to determine phases for the native data. Refinement of the structure has been initiated with 2 A resolution data.

The ferredoxins are low molecular weight iron-sulfur proteins that function as electron transfer agents in some important biological reactions. They can be divided into two main classes: the bacterial, usually with 8 iron atoms and 8 labile sulfur atoms and molecular weights usually about 6000, and the chloroplast with 2 iron atoms and 2 labile sulfur atoms and molecular weights approximately 12,000. The bacterial ferredoxins found in anaer- obes play a key role in fermentative metabolism and in nitrogen fixation, and chloroplast ferredoxins appear to function as a primary electron acceptor from the photoreactive pigments.

A preliminary report of the structure of the Fe-S complexes in ferredoxin from Peptococcus aerogenes (formerly Micrococcus aerogenes) has appeared (1). The present account describes important details of the work and provides a view of the molecule as deduced from an electron density map at 2.8 A resolution. Details of the iron-sulfur complexes that have emerged from the initial refinement of the iron and sulfur parameters by least squares based on 2,4 resolution data are also included.

EXPERIMENTAL PROCEDURE

Purification and Crystallization

P. aerogenes was grown anaerobically at 37” for 18 hours in loo-liter batches. The medium contained Difco peptone, yeast

* This work was supported by United States Public Health Service Grant GM-13366 from the National Institutes of Health.

hydrolysate, sodium glutamate, and K,HPO( to maintain the pH at 7.0 (2,3).

Previously frozen cells lyse sufficiently when suspended in distilled water for essentially only ferredoxin and rubredoxin to be released through the cell wall. After stirring the cell slurry for 1 hour, it was centrifuged at 10,000 x g and the red-brown supernatant saved. Purification was similar to previously described procedures for other bacterial ferredoxins (4, 5).

Ferredoxin is quite unstable in the presence of molecular oxygen, elevated temperatures, and pH below 6.5. In the purification and crystallization steps, therefore, it was necessary to minimize the effects of these factors. The ferredoxin solution, 0.1 M in Cl- , was concentrated by passing it over a small DEAE-cellu- lose bed just sufficient to bind all of the protein. The ferredoxin was then eluted with as small a volume as possible of 0.7 M Tris- HCl at pH 7.5, thus ensuring adequate buffer capacity for subsequent salting out with (NH&S04. The high concentration of Tris-HCl was necessary to maintain the pH at 7.5 as t,he (IVHJ2- SOc concentration was increased. Solid (NH&SO4 was added to the ferredoxin solution in order to reduce the exposure to oxygen and minimize dilution. The (NHJzS04 concentration m-as quickly taken to 2.0 M by adding solid material while the ferredoxin solution was chilled in an ice bath. The precipitate which usually formed was centrifuged at 4’ and the pellet of deteriorated ferredoxin, usually a tan-brown color, was discarded. The coffee-colored supernatant was retained and more solid (NH.& SOc added to take the concentration to 2.4 M. The solution was allowed to stand for about 5 min in an ice bath to allow a precipitate to form and the solution was again centrifuged. ,4 smaller amount of precipitate usually came down composed of the re- maining deteriorated ferredoxin along with a small amount of intact ferredoxin, giving a dark brown precipitate. If the precipitate was very dark, it was predominantly ferredoxin, and in that case the precipitate was recycled.

The following crystallization procedure was designed specif- ically for ferredoxin but has been used successfully with other proteins as well.

The concentrated ferredoxin solution was transferred to small dishes in 0.25-ml aliquots. These dishes were “cut down” shell vials of 0.4-ml capacity with ground glass lips. To the lip of each vial silicone grease was applied , giving an airtight seal when the cover slip was pressed in place. A lo- to 20-mg crystal of (NH,)zS04 was added to the solution and the region at the surface of the dissolving (NH&SO4 crystal was observed under a microscope for turbidity. Solid (NHJzS04 was added until the turbidity was observed to persist, usually at a concentration in the range 3.3 to 3.5 M (NH&SOa. The dish was then covered

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and set aside at 4”. Crystals usually developed within 1 or 2 days. If no crystals formed (as observed under a microscope at 4”), approximately 5 mg of solid (NHI)$04 were added to the solution and the dish was again covered. The advantage of this procedure is that crystallization conditions are approached quickly, minimizing the possibility that a denatured ferredoxin precipitate would contribute large numbers of nucleation sites.

Ferredoxin crystals develop as black, lath-shaped needles elon- gated along c and are frequently found growing from rosette- shaped clusters. The larger crystals selected for diffraction work were of dimensions 0.1 to 0.15 mm in cross section and 0.4 to 0.5 mm in length.

Unit Cell and Space Group

The crystals are orthorhombic, space group P2i2i2r. The unit cell parameters are a = 30.52 A, b = 37.75 A, c = 39.37 A based on CuK, = 1.5418 A. Since the density of the crystals is approximately 1.4 g.cmp3, there are 4 molecules in the unit cell, 1 per asymmetric unit (6).

Heavy Atom Derivatives

Heavy atom derivatives that finally proved successful were obtained by the usual crystal-soaking technique. Some chemical modification and recrystallization experiments were done, but none gave useful derivatives.

The nature of ferredoxin in general, and this crystal form in particular, caused great difficulty in finding useful heavy atom reagents. The sensitivity to molecular oxygen made it necessary to keep oxygen exposure to a minimum. No sulfhydryl reagent could be used because of the affinity of the labile sulfur for the reagent. The small solvent content of this crystalline form, as shown by the small V, of 1.9 (7), limited the size of ions that could diffuse into the crystalline lattice.

It was found, in fact, that few reagents caused any intensity changes whatsoever, indicating either no binding or no penetra- tion into the crystals. Those reagents that caused intensity changes were composed of small ions, but they tended to damage the crystals. Part of the damage, however, was probably the result of the extended soaking times used in the derivative at- tempts.

Success was finally achieved by “driving in” small ions or complexes at high concentrations for relatively short periods of time. Three reagents, UOz(NO&, Sm(N0J3, and PrC13 were found to give intensity changes in the x-ray diffraction pattern after soaking the crystals in 0.1 M UO zZf for 13 days and 0.04 M Sm3+ or Pr3+ for 3 davs _ .

Data Collection and Reduction

Data were collected on a computer-controlled, four-circle diffractometer in the w/20 mode at a scan rate of 2” per min in 20. Backgrounds were measured by counting for 4 s at each limit of the scan range. Friedel pairs were collected for all reflections in groups of 16 reflections: first 8 on the positive 20 side, then 16 on the negative side, 16 on the positive side, etc., so that the reflections in each pair were collected within about 15 min of each other (8). Centrosymmetric reflections were treated as repli- cates and averaged. Data were collected from a crystal until the monitor reflections had decreased in intensity by as much as 20 to 25%. Table I summarizes some statistics for each data set.

When collecting data, three monitor reflections were measured at intervals of approximately 200 reflections. For each of these reflections the ratio 1”: I, was calculated. where 1” is the initial

intensity and I, the intensity when measured as the nth reflection. Scale factors to correct each reflection for deterioration were determined from a plot of (lo/l, ) versus n. In addition, a larger set of standard reflections was measured at intervals of approximately 1500 reflections and used in the same way as the monitor reflections to determine scale factors to correct for deterioration. Agreement between the two determinations of deterioration for each crystal was satisfactory. The rate of deterioration for crystals of the native protein was approximately 3% per 1000 reflections.

Reproducibility of the observations is illustrated by the CuK, data for the native protein. Two crystals were used: one for the data from m - 2.5 A, the other for data from 2.8 - 2.0 A and from m - 5 A. Dat,a from m - 5 A were used to put the two sets on the same scale. For reflections in common to the two sets between 2.8 and 2.5 A, the relative average deviation from the mean, D, as defined in Table I, was 0.02. After collecting the data from 2.8 - 2.0 A for the second crystal, the first 147 reflections in the same shell were replicated. For the replicated set, D was 0.044.

Empirical absorption corrections were made similar to the procedure of North et al. (9) by measuring the intensity of an 001 reflection at x = 90” as a function of 4. A representative curve is shown in Fig. 1.

Lorentz and polarization factors were applied, and the structure factors computed. Those reflections where F2 < 2arz were so coded and, although retained in the data file, generally were not used.

Scaling of derivative to native data was done by the method of Singh and Ramaseshan (10). An artificial temperature factor was applied to the derivative data in order to match the falloff in intensity with increasing sin e/x to that of the native data.

Solution of Structure

The structure was solved independently with phases determined from each of three isomorphous derivatives by use of anomalous scattering measurements (8,11-13). The derivatives were subsequently combined to improve the phases used in the electron density map. A description of the various steps follow-s.

Coeficients-Coefficients representing the heavy atom vectors were computed according to the expression

p = F$ + Fb - 2.FP.FPR[l - (wk(& - F&)/2Fp)q1~2

in which k, the ratio of real to imaginary scattering, was obtained

TABLE I

Statistics for data sets

Crystal

Native. Native. Native. UO?+ deriva-

tive Sm3+ deriva-

tive Pr3+ deriva-

tive

I

l-

iadia- tion

CL1 CL1 co

co

co

co

&nin

A

2.5 2.8-2.0

2.5

3700 5200 3500

I Lxtent d’ eterio- 1 ration

%

20 20 20

0.048 0.067 0.052

3.0 2300 25 0.019

2.5 3600 23 0.05

3.0 2300 30 0.048

Da

1.078

1.036

1.03

number of replicated or svmmetrv related reflections observed.


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ods might be more powerful than the usual Fourier methods if a suitable single weight vector peak could be identified or one correct heavy atom site found. Simpson et al. (15) have shown that the symmetry map, computed by taking for each point z, y , z the minimum value of the vector map at the corresponding point on each Harker section, gives the locus of all points in the unit cell which could be interpreted as atomic sites consistent with bhe Harker sections. Such a map m-as generated on a coarse grid and showed, along with several smaller peaks, three large peaks (all of which later proved to correspond to heavy atom sites), two of which appeared in special positions in the symmetry map. The largest peak with the most general coordinates was chosen as a suitable site for atomic superposition. The Patterson origin was shifted to it and its three symmetry related positions, and the minimum function computed. From the resulting map five additional sites, all consistent with the symmetry map, were found.

One cycle of refinement of positions and occupancy (thermal parameters were fixed at 25 AZ) gave for the conventional residual, R (= Z I(FoI - /F,/I/ZIF,I), a value of 0.50 for the 3 A resolution data. Five more sites, also consistent with the superposition maps, were chosen from a difference Fourier map phased with the initial six sites. The R after refining x , y ,z, B, and m for the 11-site model was 0.44. The relative occupancies, m, were 1.3, 0.9, 1.3, 0.6, 0.5, 0.6, 0.5, 0.6, 0.3, 0.4, and 0.2. Once an approximate over-all scale was found, it was kept fixed and occupancies refined, with arbitrarily chosen scattering factors for all heavy atoms. These sites were used in calculating phases for native data by the method of single isomorphous replacement.

After refining the ll-site model, a “calculated” Patterson map was computed in which the coefficients used were the calculated structure factors of the heavy atoms. Comparison with the experimental Patterson map suggested that the major peaks were satisfactorily accounted for. It was discovered in a subsequent reexamination of the difference map, however, that an additional site of relative occupancy approximately 0.7 had been neglected. When this site was included, the R value was 0.39 (3 -4 data). This 12.site model was used as input for the combined phasing procedure.

Samarium Derivative-In contrast to the difference Patterson map for the UOz 2+ derivative, that for the Sm3+ derivative was readily interpreted in terms of two major sites with approximately the same occupancy and similar z coordinates. The R value for the two-site model was 0.47 for the 2.5 A resolution Co data. A difference Fourier map showed two additional sites at about one- third the occupancy of the first two. The R value after refinement of the four-site model was 0.44. An approximate calculation by two methods of the absolute occupancy of the Sm3+ indicated that the major sites corresponded to 25 to 28 electrons, i.e. approximately 0.4 to 0.5 occupied. The four-site model was used for computing the phases based on this derivative alone.

FIG. 1. Typical absorption curve for ferredoxin. Inset shows cross-section of crystal in capillary; angular label corresponds to abscissa of diagram.

experimentally from a plot of the ratio of the isomorphous differences to anomalous differences as a function of sin20/X2 (14).

Patterson maps computed with w = 1.0, where w is a weighting factor to account for the relative accuracies of anomalous versus isomorphous measurements, rather than 0.75 suggested by Matthew (14), seemed sharper than those using the latter value. The possibility of the difference in the phases of the heavy atom vectors and that of the protein being greater than 90” was not taken into account, i.e. the positive root of the bracketed term was always used. The rationale was that in the few cases where it would be negative, less error would be made than if it were mistakenly taken as negative when, in fact, it should have been taken positive. The coefficients for d spacings greater than 10 A were not included since they are most sensitive to changes in the solution.

Inasmuch as these coefficients represent the heavy atom vector magnitudes, they were used as “observed” structure factors for conventional least squares refinements and difference Fourier maps in searching for additional sites.

Choice of Radiation-Initially, data had been collected with CuK, data from two UO$f derivatives. The first one proved to be lightly substituted and gave an uninterpretable difference Patterson map. A second derivative, more heavily substituted, gave a similar map. Reasoning that the considerable anomalous scattering from the iron atoms in both native and derivative crystals was, in part, responsible for the complicated maps, we decided to use a radiation such as Cr or Co in an effort to improve them. Absorption was so serious for Cr radiation, however, that the usual empirical correction was of questionable validity, but Co data could be corrected with reasonable confidence. Accord- ingly, sets of Co data were collected from a native crystal and a third UO@ derivative crystal. The difference Patterson map was again complicated, suggesting multiple sites, but it was interpretable using a systematic approach.

Uranyl Derivative-Few prominent peaks in the Harker sections gave consistent sets of coordinates for heavy atom sites, indicating a multiple site derivative in which non-Harker cross vector peaks were as large as or larger than Harker peaks from symmetry-related atoms. It appeared that superposition meth-

Before going on to the combined phasing procedures, we reexamined the difference map and an additional six minor sites were included giving an R of 0.41. Another difference map suggested two additional sites. The 12-site model after refinement gave an R of 0.39 (2.5 A data). “Calculated” Patterson maps computed at various stages confirmed the improvement obtained in R by the additional sites. The 12-site model was used as input for the combined phasing procedures.

Praseodymium Derivative-The difference Patterson for the Pr3+ derivative was nearly identical to the one for the Sm3+ derivative, and use of the coordinates of three of the initial four Sm3+ sites gave an R of 0.48 for the 3 A resolution Co data. A


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TABLE II Statistics for single isomorphous derivative phasin.g

Derivative No. of sites No. of phases <m>” RP RKC Red

%

uo2*+.......................... 11 731 0.61 8.8 12.9 48.8 Sm3+. 4 1115 0.64 5.5 8.2 47.8 P?+. 8 760 0.63 5.0

I I 7.1 47.1

a (m) = mean figure of merit = (cos 01). bRl = xhkt j / FpH I - I FP + fx I j / CMC I Fp~ I X 100 over all data. c RK = &C 1 1 FpH / - j Fp + f~ 11 / CJ,~C / FpH 1 X 100 over centrosymmetric data.

- zBkl 1 I FpH ( - 1 Fp + fH 1 j / CM 1 1 FPH I - I FP 11 X 100 over centrosymmetric data. : fit: = [z (I FPH 1 - 1 FP + fH 1)*/n]"".

f E, = 1 c [I F$H I - 1 FFH I + 2(l FP 111 FPX I)W”l(fo + &‘)I @ ~0s a - a ~0s ~)12/41’2.

<Ec>~ <E,>f

34.9 18.3 18.9 14.7 19.0 15.5

difference map phased with these showed five more sites which reduced R to 0.43. The eight-site model was used for computing phases based on this derivative alone.

Before going on to the combined phasing procedure, the difference map was reexamined and two additional sites added which were close to one of the major sites. Including the two additional sites reduced R to 0.41 (3 A data). A AF map indicated no more sites with relative occupancies greater than 0.1. Six or seven of the Pr3+ sites are close to or identical with sites in the Sm3+ derivative. The lo-site Pr3+ model was used as input for the combined phasing procedures.

Single Isomorphous Derivative Phasing-Initially, all derivatives were treated independently as a check that the structure found was, in fact, the correct one. Thus, two maps were computed for each derivative, corresponding to the two enantio- morphs for the heavy atom sites. The relevent statistics for the phasing are summarized in Table II.

The Sm3f phased maps were computed first, one of the two appeared to be incorrect with no recognizable protein features, while the other revealed most of the structure (1). Two regions of outstanding electron density were obvious and were interpreted in terms of 4 iron and 4 sulfur atoms approximately at the corners of a cube with 4 cysteine sulfur atoms coordinated to each iron as found in high potential iron protein from Chroma- i&m (16). The two clusters thus account for the 8 iron, 8 labile sulfur, and 8 cysteine sulfur atoms in this ferredoxin. As expected, the PI?+ phased map was similar to that based on Sm3+, although it was of poorer quality because the resolution was lower and the Pr3f derivative deteriorated more rapidly in the x-ray beam.

The UO,* phased map proved to have been referred to a different origin, related to the Sm3+ phased map by a translation of fh along 2, a fact which was readily established by inspecting the positions of the iron-sulfur clusters relative to each other and to the symmetry elements. Although the course of the polypeptide chain could be followed through most of the molecule with little difficulty in the 2.5 A Sm3+ phased map, it was decided to calculate a Fourier map based on phases from the combined derivatives before making a detailed interpretation.

Combined Phasing and 2.8 A Resolution Electron Density Map-The 12-site UOs2+, the 12-site Sm3+, and the lo-site Pr3+ model were used together in three cycles of alternate phasing and refinement, minimizing the closure error by refining scale, positions, and occupancy (17). The resultant figure of merit was 0.726, reflecting in part the improvement gained by combining the derivatives and, in part, the inadvertant limiting of the data to 2.8 A. The residual R for the UOz2+ derivative apparently improved at the expense of that for the Pr3+ derivative.

TABLE III

Combined phasing rejinement criteria

TABLE IV

Heavy atom sites from combined phasing in fractional coordinate and relative occupancies

Sites are grouped according to proximity and may not be ex- actly the same for the three derivatives. Superscripts have the following meaning: 1, site used in single isomorphous replacement method; 2, site found by difference synthesis using Matthew’s coefficients; 3, site found by conventional difference map; 4, site found in superposition map (UO 22+ derivative only) ; 5, site found in symmetry map (UOz2+ derivative only).

; 0.845’l3 0.85Z1 0.263 0.222

* -0.012 0.015 DCEup 0.58 I 0.22

c o.3401,4,5 o.*865 0.143 0.130

L&p 0.76 0.049 0.031 0.10

x 0.520' 0.60Z3 I 0.280 0.307

c& 0.303 0.86 0.290 0.19

3

Pf’

0.149’ 0.228 0.093 0.75

0.877’ 0.362 0.040 0.97

0.864’ 0.269 0.019 0.26

0.3962 0.007 0.141 0.34

0.2693 0.142 0.043 0.15

0.5901 0.306 0.292 0.27

SP

0. m4* 0.005 0.200 0.18

0.857’ 0.296 0.215 0.12

0.0813 0.176 0.029 0.04

0.222 0.235 0.100 0.09

*c+’

The root mean square isomorphous and anomalous closure errors were allowed to change in each cycle since they appeared to be converging without undue interaction. Closure errors changed by about 10% and scales by about 1%.

In order to assess the validity of the minor sites and because the statistics for Pr3+ were relatively poor, difference Fourier (14) maps were calculated for each derivative using the phases of the protein data from the last cycle of refinement. On the basis of these difference maps, six of the 12 sites for the U022f


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derivative were omitted and a new one was added; for the Sm3+ derivative, three sit.es were omitted and three new ones added; for the IV+ derivative, three sites were omitted and three new ones added. The revised coordinates were included in a new series of phasing and refinement. After two cycles, all seven UO$f sites behaved well, three low occupancy Sm3f sites were omitted (two of the new ones) and one was reinstated, and three PrY+ sites were eliminated. One additional cycle of refinement and phasing was carried out. Statistics for the final refinement are given in Table III and the final refined parameters in Table IV. Fig. 2 shows the refinement criteria as a function of sin28/X2. Although convergence may not have been complete, refinement was terminated since there appeared to be little significant change in either coordinates or occupancy.

Centroid phases were used in calculating an electron density map at 2.8 A resolution with coefficients weighted by their figure of merit. The map was calculated on a grid a/72, b/76, c/78 so that the print-out from the computer was on the same scale as the Kendrew protein models. Fig. 3 is a composite of 2, y sections from the map. Each view is composed of six sections and represents the electron density in successive 2.52 A thick slices through the molecule.

140

120

100

60 IFI

60

40

20

140

I20

100

60

6.0 A 4.0 h 3.5 A 3.0 A 2.7A 2.5 A

‘------ a

O-----Y IFPl/Z

\ NAT.

.\

&-A -----A m l --b ---.\., .A... --._ .

.Ol .02 .03 .04

sin%/,:

60i 4.06 3.5 i 3oA 2.7; 2.5i

b

SM’E

E. O--o------

.Ol .02 .03 .04

sin%/<

1.0

0.6 m

0.6

0.4

0.2

1

1

3991

Model of Structure

Fitting the Model-The map was relatively clean and the course >f the main chain could be readily followed. Most of the R groups were evident and the two tyrosine and some of the pro- ine groups could be identified. For a detailed interpretation If the map, however, use of the sequence was essential (Fig. 4) cl@. The sense of the chain was determined as soon as the mique sequence of 2,2,3 and 2,2,3 residues between the cys- seine groups could be identified. Since the molecule has internal symmetry (la), the chain termini could have been confused with the midpoint of the chain in view of the low main chain density in the region of Residue 27. This possibility was ob- viated, however, because the proline residues unique to each nalf of the molecule could be readily identified, particularly Pro 16, Pro 50, and Pro 52.

Kendrew models of the main chain and side chains were fit to the electron density in a Richards optical comparator (19). Several parts of the molecule proved difficult to fit, however, and the loops of chain with two residues between cysteine groups were especially difficult.

The iron-sulfur complexes labeled I and II in Fig. 3 were fit by a model constructed of 3&-inch bronze rod on a scale 2 cm

140

120

100

60 IFI

60

40

140

120

too

60 IFI

60

40

20

6.OOA 4.oA 3.5 A 3.0 b

C

PR”

01 .02 .03

sit&/P

6.0 A 4.0 A 3.5A 3.0 A

d

AFI A

\

IJo;=

\ +----h___\

E’ “\AeA-A IAF.IO--___ o-o-.

Ea O----~-o ---0

.a .02 .03

sin%/,:

FIG. 2. Statistics for native ferredoxin crystals and three derivatives plott,ed as a function of sin2 B/X2. a, 1 F, 1 is average magnitude of structure amplitudes, m is figure of merit. b, c, and d, 1 AF; 1 is r.m.s. difference between amplitudes of heavy atom derivative and native crystals; 1 AF, 1 is r.m.s. difference between 1 Fnkl I and / FluEi 1; Ei and E, are r.m.s. lack of closure errors which apply to I AF; j and / AF, 1 .


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b 0-

r ’

FIG.

3.

El

ectro

n de

nsity

m

ap

at

2.8-

A re

solu

tion.

Ph

ases

ba

sed

on

com

bine

d da

ta

for

UOn*

+,

Sm3+

, an

d PI

,3+

to

3-A

reso

lutio

n an

d on

da

ta

for

Sm3+

fro

m

3 -

2.8

A re

solu-

tio

n.

Dire

ctio

n of

vie

w is

al

ong

-c:

cont

ours

at

eq

ual

arbit

rary

in

terv

als

of

elec

tron

dens

ity.

Each

vie

w is

a

com

posit

e of

six

x,y

se

ctio

ns

and

repr

esen

ts th

e el

ectro

n de

nsity

in

a

slic

e of

th

e m

olec

ule

2.52

A

thick

. CI

, 54

/78

- 40

/78,

b,

40

/78

- 44

/78,

c,

44

/78

- 39

/78,

d,

30

/78

- 34

/78,

e,

34

/78

- 29

/7&f

, 29

1’78

-

24/7

& ~1

,2-1

/78

- 19

/78,

h,

N/

78

- I4

/ 78

, i,

14/7

8 -

O/78

, j,

O/78

-

4/78

.

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3993

I 5 IO 20 25

30 35

FIG. 4. Sequence of Peptococcus aerogenes ferredoxin (18). doxins compared in Reference 24.

I Fe-Fe:3.258 S-S:3.258 \

Fe-Fe:2.85(.0S)J(

s-S:3.53(.10)A

a b

FIG. 5. Model of the iron-sulfur complex. a, used to fit the 2.8-A resolution electron density map; b, from initial least squares refinement of the complexes. Numbers in parentheses after bond lengths and angles are estimated standard deviations of the mean vallles based on the sample variance. Values are X2 to allow for the preliminary nature of refinement

40 45 50 54 Invariant residues, :; semiinvariant, 9 ; based on five clostridial ferre-

Structure Factor Calculations and Initial Refinement--Atomic positions from the model were marked by adhesive dots on the plastic sheets of the three-dimensional electron density map. It can be seen on inspecting Fig. 3 that in a number of places atoms are outside the lowest contour in the map. The fit could have been improved by distorting the bond angles of the model, but we did not wish to physically alter them. Instead, when the atomic positions on the plastic sheets were transferred to the corresponding sections of the computer print-out, they were adjusted to better fit the electron density. Coordinates were estimated to 0.1 grid spacing directly from the print-out. Pa- rameters for the C, atoms and the iron and sulfur atoms are listed in Table V.

In the first structure factor calculation, the two oxygen atoms in the Glu 17 R group were omitted because of uncertainty in their positions and 22 water oxygen at,oms were included, a

36 54

6

FIG. 6. plot of the cy carbon, iron, and sulfur positions. Direction of view along -c. Fe, 0 ; Sinarg, 0 ; SW, @ ; Cm, l

A-l. Iron and sulfur atoms are at alternate corners of a cube total of 410 atoms in the asymmetric unit. With an over-all 2.3 A on an edge and cysteine sulfur atoms are on the extended B of 10 A2, t.he residual R w-as 0.449 for the 2904 reflections in cube diagonals through each iron atom. Fig. 5a shows the model the range 0.05 < sin 0/x < 0.25 (2-A data) and greater than of the complex used. 2ar. The 32 reflections with sin 0/x < 0.05 were given zero


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3994

TABLE V

Coordinates of C, atoms from 2.8 A resolution electron density map; coordinates of sulfur and iron atoms from initial resnement;

Cal Ca2 Ca3 Ca4 Ca5 Ca6 Ca7 Ca8 Ca9 Cal0 Ca 11 Cal2 Ca 13 ca14 Cal5 Cal6 Cal7 ca 18 Cal9 Ca 24 Ca21 ca 22 Ca23 ca24 Ca25 Ca26 Car27 Ca28 Ca 29 Ca30 Ca31 Ca32 Ca33 ca34 Ca35 Ca36 Ca37 Ca38 Ca39 Ca40 Ca41 ca42 ca43 ca44 Ca45 Ca46 ca47 ca48 Ca49 Ca50 ca51 ca52 ca53 ca54

x Y

18.2 h -1.1 A 20.5 1.0 21.8 1.7 24.7 4.4 24.7 6.3 27.1 8.6 27.2 11.3 29.7 8.9 32.7 11.2 34.8 8.1 36.1 9.9 36.2 7.6 34.7 7.8 31.4 6.6 32.5 3.0 32.7 3.8 29.6 5.1 28.4 1.9 27.2 0.3 26.8 -2.3 30.0 -2.3 28.7 -3.0 30.8 -1.0 32.4 -1.5 34.7 -0.2 35.4 1.2 32.9 3.5 31.1 2.4 28.7 0.2 26.8 -1.5 25.3 -4.4 22.4 -5.2 22.4 -8.3 23.4 -6.1 22.0 -2.7 19.1 -1.7 19.2 1.5 20.4 3.6 20.5 6.7 23.7 7.0 26.1 7.7 23.9 9.3 24.4 12.2 27.3 12.5 27.7 12.2 28.3 14.9 27.2 14.7 24.5 13.2 24.6 10.1 23.1 6.9 20.1 6.0 17.6 4.4 16.9 3.5 17.3 -0.2

15.4 A 13.3 10.1

9.1 5.8 4.3 7.0 a.3 8.6 9.6

12.6 14.1 17.2 18.8 18.9 22.7 24.7 22.9 26.3 23.7 21.9 18.3 16.1 13.1 11.3

8.0 6.6

10.1 11.3 14.5 13.4 15.5 17.7 20.4 19.6 22.2 20.7 23.2 21.2 21.7 19.7 17.7 20.2 18.8 15.4 13.8 10.5 11.9 10.2 12.6 12.4 13.6 10.6 10.6

s a7 30.87 6.87 9.75 Slly 33.29 10.79 14.56 s14y 30.42 5.66 16.07 slay 27.13 0.26 21.10 s35y 21.30 -0.62 18.16 ssay 22.53 3.23 23.04 5417 24.39 5.19 17.62 s45y 27.64 11.16 13.04 S123 32.06 7.15 13.38 8124 30.51 10.25 11.88 s134 28.61 7.30 12.73 S234 30.00 9.72 15.12 S567 23.87 -0.23 20.79 S568 25.12 1.26 18.03

s578 25.31 2.94 20.75 S678 22.14 2.62 19.33 me 8-1 30.41 7.88 11.50 Fell-2 31.72 9.50 13.42 Pe14-3 30.32 7.21 14.20 &45-4 29.12 9.65 13.09 PelS-5 25.55 0.93 20.29 Fe35-6 22.82 0.56 19.05 m38-7 23.51 2.15 21.50 Fe41-8 24.12 3.11 la.77

-coordinates in Angstroms

z

weight because the solvent continuum was omitted from the model (20).

Considerably improved positions of the iron and sulfur atoms

in the complexes should result from least squares refinement of the parameters for these atoms while keeping the rest of the atoms fixed. Refining in this way should work reasonably well with Z-A data since the iron and sulfur atoms are separated by distances which considerably exceed this value. Accordingly, four least squares cycles were calculated in which 1% , y , z and B for the 24 iron and sulfur atoms were varied. The residual R decreased to 0.405.

RESULTS

De.scri@ion of Structure-The molecule can be described as a prolate ellipsoid with approximate dimensions 22 A x 27 A, the major axis being essentially parallel to the line joining the centers of the two iron-sulfur complexes. The complexes arc the most prominent feature in the molecule, lying within it and separated by a distance of about 12 A. For the most part, they are covered by a layer of main chain or by side chains. The C, atoms along with the iron and sulfur atoms are shown in Fig. 6 viewed in the same direction as the composite of sections in Fig. 3.

The two iron-sulfur complexes appear to be identical, each containing 4 iron and 4 inorganic sulfur atoms with 4 cysteine sulfur atoms coordinated to t’he iron atoms. The iron and ill- organic sulfur atoms in each complex are arranged in approximate cubes with iron and sulfur atoms at alternate corners of each face. The initial refinement has shown that the central cube-like regions of the complexes are considerably distorted (Fig. 56) and better described in terms of interpenetrating tetrahedra, one with iron atoms at the vertices, the other with sulfur atoms at the vertices. The tetrahedra appear to be regular and to have a common center, the iron ones are approximately 2.8 A on an edge, the sulfur ones approximately 3.5 A on an edge. The bond lengths and angles in Fig. 5b are average values for the two complexes based on the assumption of T - 23 symmetry. At the present stage of the structure analysis, the complexes in ferredoxin do not differ significant#ly from the one in high potential iron protein (16) or the Fe,& complex in the compound (Et4N)2[Fe&(SCHzPh)41 (21). It is now clear, however, that the Fe& part of the complexes is not the same as the ones in two cyclopentadienyl iron sulfide structures which have been reported (22, 23).

In the sequence (Fig. 4) (18), there are two groups of 4 Cys residues: Cys 8-11-14-B and Cys 35-38-41-45, both with 2,2,3 residues between the cysteines. These two groups are not in- corporated in the clusters, however, as might have been expected. Fig. 6 shows, instead, that Cys 8, 11, 14, and 45 are coordinated in one complex (Complex I) and Cys 18, 35, 38, and 41 in the other (Ccmplex II). It will also be seen that it is the two 3-residue sections between Cys 14 and Cys 18 and between Cys 41 and Cys 45 that serve to join the two complexes. The possibility for symmetry in this arrangement is evident in the schematic diagram (Fig. 7) where an approximate a-fold axis is

FIG. 7. Schematic diagram showing approximate a-fold symmetry.


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FIG. S. Plot of the cy carbon, iron, and sulfur positions showing approximate 2-fold symmetry.

present. In fact, in the structure an approximate 2-fold axis relates the first half of the molecule1 to the second half with the NH2- and COOH-termini of the chains in close proximity (Fig.

8). The aromatic groups present in the molecule, Tyr 2 and Tyr 28,

are each oriented in a similar way with respect to one of the complexes. Tyr 2 is parallel to and apparently in contact with one “face” of the central high density region of Complex II and similarly for Tyr 28 with respect to Complex I.

Secondary Structure-It is not surprising that secondary structure of the helical or pleated sheet type is minimal in view of the short protein chain involved in coordinating to 8 iron atoms in t,he two complexes.

TABLE VI

Environment of complexes

, Face

- Complex

I

II

Residues “seen”

S-11-14 s-11-45 8 - 14 - 45

11 - 14 - 45

35 - 3s - 41 35 - 3s - 1s 35 - 41 - 1s 3s - 41 - 1s

Tyr 28, Gly 12 Ile 9, Val 47 Ala 49, Ile 4, (Pro 50) Val 44, Ala 13, Pro 46

I -

Tyr 2, Gly 39 Ile 36, Val 20 Ile 22, Ile 30 (Pro 19)

this is a very short section of antiparallel pleated sheet in which the two terminal sections of the chain are hydrogen bonded together. In addition, 037 appears to be hydrogen bonded to the NHz-terminal amino group anchoring it to the body of the molecule.

A prominent interaction between main chains involves the hydrogen bonds between N3.. ,051 and 03. . .N50. Indeed,

side chains virtually cover the complexes on all sides. Table VI lists such groups at each face of the tetrahedra formed by the cysteine sulfur atoms.

Cys 8, 11, and 45 in Complex I and Cys 38 and 41 in Complex II appear to be somewhat exposed at or near the surface of the

Most of these groups are either invariant

molecule, although they tend to be shielded by the main chain

or conservatively replaced in the clostridial ferredoxins for which

or surrounding side chains.

the sequences are known (24).

36

There may also be some other hydrogen bonds corresponding to a very limited pleated sheet or 310 helix, but they can only be regarded as tentative at this stage of the structure analysis. There are a number of residues where 0, appears to be directed toward the general region of Nn+2 and some of these may correspond to a 27 ribbon.

Environment of Complexes-The folding of the chain beyond the requirement of cysteine groups in the proper positions to coordinate to the iron atoms appears to be a result of packing hydrophobic residues around the complexes. Hydrophobic

1 1. Birktoft, personal communication.

Fig. 9 demonstrates common features of the complexes when they are oriented in the same way. The sections of chain containing three cysteines near one another in each half of the molecule are extended in such a way that side groups protrude in alternate directions. The cysteines are thus in the proper positions for bonding in each complex and the hydrophobic residues near the two complexes are oriented similarly. The invariant glycines are also seen to occur in the same relative positions


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3996

COMPLEX I COMPLEX II

FIG. 9. Schematic representation of side chains in the complexes which are the same in the two halves of the molecule.

and as not,ed above each tyrosine is oriented in a similar way with respect to one of the complexes.

DISCUSSION

Invariant Residues-The invariant amino acids in the five known sequences (24) involve 26 of t,he 54 in P. aerogenes. It would not be surprising if the invariant residues tended to be concentrated in the vicinity of the complexes and this is, indeed, the case. Consider the two groups of 11 residues involving the two groups of 4 Cys residues along with the 4 residues on either side of the two groups, i.e. residues 4 to 22 and 31 to 49. These two groups account for 38 of the 54 amino acids and 24 of the 26 invariant residues are within them. Only two of the remain- ing 16 amino acids are invariant, the NHz-terminal Ala and Gly 25.

(1962) B&hem. Biophys. Res.‘ Comm&. 7, 448 5. LO~ENBERG, W., BUC&A&AN, B. B., AND RABIN• WITZ, J. C.

(1963) J. Biol. Chem. 236, 3899 6.

7. 8.

9.

10.

11.

SI&ER; L. C., AND JENSEN, L. H. (1965) Biochem. Biophys. Res. Commun 20, 33

MATTHEWS, B. W. (1968) J. Mol. Biol. 33, 491 HERRIOTT, J. R., SIEKER, L. C., JENSEN, L. H., AND LOVEN-

BERG, W. (1970) J. Mol. BioZ. 60, 391 NORTH, A. C. T., PHILLIPS, D. C., AND MATHEWS, F. S. (1968)

Acta Crystallogr. Sect. A 24, 351 SINGH, A. K., BND B~MASESHAN, S. (1966) Acta CrystaZZogr.

21, 279 BLOW, D. M., AND ROSSMANN, M. G. (1961) Acta Crystallogr.

The invariant Gly 12 and Gly 39 along with Gly 25 appear to enable Tyr 2 and Tyr 28 to be aligned next to a complex. ,4sp 37 is invariant, and as noted above, it serves to hydrogen bond the NHz terminus to the body of the molecule.

14, 1195 12. NORTH, A. C. T. (1965) Acta Crystallogr. 18, 212 13. MATTHEWS, B. W. (1966) Acta CrystaZZogr. 20, 82 14. MATTHEWS, B. W. (1966) Acta Cr2/staZZo&. 20, 230 15. SIMPSON. P. G.. DABBOTT. R. D.. AND LIPSCOMB. W. N. (1965)

Nature of Functional Groups-There can be no doubt that the iron and sulfur complexes are involved as the active centers in ferredoxin, but the similarity in location of the two aromatic residues suggests that they play a role in transferring the electrons. Both are mostly buried in the protein, but with an edge exposed to the solvent. Furthermore, as noted above, the plane of each ring is parallel to and essentially in contact with one face of the central high density region of one complex. Although Tyr 2 and 28 are not invariant, they are aromatic in all the available ferredoxin sequences (24)) and thus would be expected to pack in the molecule in the same way as the two aromatic groups in P. aerogenes. It is interesting that a recent observa- tion on the 2Fe-2S protein, adrenodoxin, ascribes an anomalous emission to a tyrosine in that protein, possibly as the result of formation of an “exciplex” with some unknown moiety such as the iron-sulfur chromophore (25).

16.

17.

18.

Acta &ystaZlbgr. 18, 166 CARTER, C. W., FREER, S. T., XUONG, Ng. H., ALDEN, R. A.,

AND KRAUT, J. (1972) Cold Spring Harbor Symp. Quant. BioZ. 36, 381

DICKERSON, R. E., WEINZIERL, J. E., AND PALMER, R. A. (1968) Acta Crystallogr. Sect. B B24, 997

TSUNODA, J. N., Y~SUNOBU, K. T., AND WHITB:LEY, H. R.

19. 20.

21.

(1968) j. BioZ.‘Chem. 243, 6262 RICHARDS. F. M. (1968) J. Mol. BioZ. 37. 225 WATENPAkGH, K.‘D., ‘SIEICER, L. C., HERRIOTT, J. I%., AND

JBNSI~N, L. H. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 359

22.

HERSKOVITZ, T., AVERILL, B. A., HOLM, R. H., IBERS, J. A., PHILLIPS, W. D., AND WEIHER, J. F. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 2437

SHUNN, R. A., FRITCHIE, C. J., AND PREWITT, C. T. (1966) Inorgan. Chem. 6, 892

23.

24. The similarity of the iron-sulfur complexes in ferredoxin and

in high potential iron protein is of particular interest in view of the very different E. values of the two proteins, -0.4 V and

WEI, C. H., WILKES, G. R., TRF,ICHEL, P. M., AND D.~HL, L. F. (1966) Znorgan. Chem. 6, 900

TANAKA, M., H~NIU, M., MATSUEDA, G., YASUNOBU, K. T., HIM~S. Ii. H.. AIC~GI. J. M..B~RNRs.E. M.. AND DEVANATHSN.

25 T. (19jl) J. ‘Biol. &em. i46, 3953’ ’

KIMUR.4, M., AND TING, J. (1971) Biochem. Biophys. Res. Commun. 46, 1227

+0.35 V. respectively. Presumably, the large difference in 26. EISENSTEIN, K. K., AND WANG, J. H. (1960) J. BioZ. Chem.

(26) that there are two independent redox centers, each capable of one electron transfer.

Acknowledgments-The computer programs used for this work include the X-RAY 70 System (27) and ORTEP (28). The phasing program used was originally provided by Dr. R. E. Dickerson and subsequently modified by Drs. J. R. Herriott and K. D. Watenpaugh. The superposition program used was written by Dr. C. E. Nordman and implemented here by Dr. J. C. Hanson. We wish to thank Dr. H. R. Whiteley for the P. aerogenes inoculum and to acknowledge the late Professor Philip E. Wilcox for his interest in this structure and for his many helpful discussions.

REFERENCES

1. SIEKER, L. C., ADMAN, E., .~ND JENSEN, L. H. (1972) Nature 236, 40

2. WHITELEY, H. R. (1952) J. Bacterial. 63, 163 3. WHITELEY, H. R. (1957) J. Bacterial. 74, 324 4. MORTENSON. L. E.. VALENTINE. R. C.. AND CARNAHAN, J. E.

, _ potential is associated with differerit. environments about the 244, 1720

iroll-sulfur complexes in the two structures.2 27. STEWART, J. M., KUNDELL, F. A., AND BI\LDWIN, J. C. (1970)

The existence of the two complexes in this ferredoxin bears Computer Science Center Report, University of Maryland

28. JOHNSON, K. C. (1965) Oak Ridge National Laboratory Report out the chemical evidence presented by Eisenstein and Wang ORNL-SYSQ, Revised

29. CARTER, C. W., JR., KROUT, J., FREER, S. T., ALDEN, R. A., 2 A more recent comparison of the complexes and a possible SIEKER, L. C., ADMAN, E., AND JENSRN, L. H. (1972) Proc.

explanation for the difference in potentials has appeared (29). Nat. Acad. Sci. U. S. A. 69, 3526


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Elinor T. Adman, Larry C. Sieker and Lyle H. JensenThe Structure of a Bacterial Ferredoxin

1973, 248:3987-3996.J. Biol. Chem.

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The Structure of a Bacterial Ferredoxin*