Proc. Natl. Acad. Sci. USAVol. 87, pp. 598-602, January 1990Biochemistry

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I:[Fe-S] cluster-driven protein rearrangementA. E. MARTfN*, B. K. BURGESS*t, C. D. STOUTS, V. L. CASH§, D. R. DEAN§, G. M. JENSEN¶,AND P. J. STEPHENS¶*Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717; tDepartment of Molecular Biology, Research Institute ofScripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037; §Department of Anaerobic Microbiology, Virginia Polytechnic Institute and StateUniversity, Blacksburg, VA 24061; and IDepartment of Chemistry, University of Southern California, Los Angeles, CA 90089-0482

Communicated by Richard H. Holm, September 18, 1989 (received for review June 8, 1989)

ABSTRACT Azotobacter vinelandii ferredoxin I is a smallprotein that contains one [4Fe-4S] cluster and one [3Fe-4S]cluster. Recently the x-ray crystal structure has been redeter-mined and thefdxA gene, which encodes the protein, has beencloned and sequenced. Here we report the site-directed muta-tion of Cys-20, which is a ligand of the [4Fe-4S] cluster in thenative protein, to alanine and the characterization of theprotein product by x-ray crystallographic and spectroscopicmethods. The data show that the mutant protein again containsone [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S]cluster obtains its fourth ligand from Cys-24, a free cysteine inthe native structure. The formation of this [4Fe-4S] clusterdrives rearrangement of the protein structure.

Nonheme iron-sulfur [Fe-S] proteins contain clusters com-posed of Fe, S2-, and cysteine residues. These proteins occurin all organisms and are central to numerous biological pro-cesses (ref. 1 and references therein). To meet such diversefunctional demands, [Fe-S] proteins have understandably hadto adopt equally diverse structures. This is evident in theirmolecular weights, which range from 6000 in bacterial ferre-doxins to 300,000 or more in multimeric enzymes, and in theexistence of multiple types of [Fe-SI clusters containing dif-ferent numbers of Fe atoms. Although it is known that thestructures, redox properties, and chemical reactivities of pro-tein bound [Fe-SI clusters are highly dependent on the asso-ciated polypeptide structure, the rules governing these inter-relationships are as yet very poorly understood (1).To elucidate the relationships between primary structure

and [Fe-SI cluster structure and reactivity, we have producedsite-directed mutant versions ofAzotobacter vinelandii ferre-doxin I (AvFdI) [nomenclature of Yoch and Arnon (2)]. Thestructure of AvFdI has been redetermined (3-5) by x-raycrystallography and the data show that AvFdI contains one[4Fe-4S] cluster, one [3Fe-4S] cluster, and two free cysteineresidues in its air-oxidized state. The [3Fe-4S]1+ undergoespH-dependent one-electron reduction at about -420 mVversus the standard hydrogen electrode (2, 6-8). The 1+/2+redox potential of the [4Fe-4S] is much lower (7). Studies ofthe analogous Azotobacter chroococcum ferredoxin I (FdI)show that its [4Fe-4S] has one of the lowest potentials (about-645 mV) so far reported for a biological [Fe-S] cluster (9).Here we report the site-directed mutation of cysteine residue20, which is a ligand of the [4Fe-4S] cluster, to an alanine andthe characterization of the mutant protein by x-raycrystallography11 and spectroscopic methods.

METHODSMutagenesis offdrA and Expression of the Altered FdI. A

mutant strain of A. vinelandii, LM100, which does not

express FdI, does not exhibit a detectable phenotype (10). Itwas therefore necessary to use indirect mutagenesis andexpression procedures to mutate thefdxA gene and expressthe altered FdI protein. This was accomplished in severalsteps. In the first step, the clonedfdxA gene (10) was isolatedand then fused to the nif structural gene cluster from A.vinelandii in plasmid pDB6, which carries the entire A.vinelandii nif structural gene cluster as well as flankingregions (11). This fusion resulted in the deletion of the entirenifW gene and the amino-coding portion of the nifD gene andplaced fdxA gene expression under the control of the nifHpromotor. The fused nif-fdx region was then cloned intoM13mpl8 and single-stranded DNA from the resultant hybridphage was used for site-directed mutagenesis of the fdxAgene (see below). Once the desired mutation was introducedinto thefdxA coding region and the sequence was confirmedby dideoxynucleotide DNA sequencing, the nif-fdx-containing DNA fragment was isolated and recloned into thehybrid plasmid (pDB6) used for the original fdx-nif genefusion construction. Plasmid DNA (pDB213) and crude wild-type A. vinelandii rifampicin-resistance genomic DNA werethen mixed (about 50 and 1.0 ,g, respectively) and added tocompetent cells ofA. vinelandii strain LM100, which containa kanamycin cartridge within thefdxA gene and is FdI- (10).Transformed cells were recovered by plating on Burk me-dium (14) supplemented with ammonium acetate (30 mM) andrifampicin (5 ,ug/ml). Strains that had recombined the alteredfdxA gene into the nifregion were subsequently identified byscoring for rifampicin-resistant transformants that were alsoNif. These strains were also kanamycin-resistant. A de-tailed description of the gene replacement procedure is foundelsewhere (12). The resultant strain (C20A; Fig. 1) expressesthe altered FdI product in response to nitrogenase derepres-sion but does not express the wild-type FdI protein. Oligo-nucleotide-directed mutagenesis was performed by themethod of Zoller and Smith (13). The oligonucleotide used formutagenesis has the sequence: TGTGTTGAAGTCGC-CCCGGTAGACTGTT. The underlined nucleotides repre-sent substitutions that change codon 21 within thefdxA genefrom TGC to GCC, resulting in the substitution of alanine forcysteine at position 20 in the FdI protein sequence. Wild-typeand the C20A mutant strain of A. vinelandii were cultivatedand derepressed as described (14). The mutant protein waspurified using the same method used to obtain single crystalsof the native protein (4) except that anaerobic conditionswere used throughout and buffers contained 1 mM Na2S204and 1 mM dithiothreitol.X-Ray Crystallography. The C20A mutant FdI was crys-

tallized in the tetragonal form suitable for x-ray crystallog-

Abbreviations: AvFdI, Azotobacter vinelandii ferredoxin I; FdI,ferredoxin I; HiPIP, high-potential iron protein.tTo whom reprint requests should be addressed.IThe atomic coordinates described here have been deposited in theProtein Data Bank at Brookhaven (accession no. 1FD2).

598

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Proc. Natl. Acad. Sci. USA 87 (1990) 599

n/f Hp -4 fdxA

Mstf]

nIf D

TApa I

- 500 bp -I

FIG. 1. A. vinelandii genomic region that contains the fdxA-nifgene fusions. The region from the nif structural gene cluster locatedbetween the Mst II and Apa I restriction enzyme sites was deletedin this construction. All of the nifH coding sequence and a portion ofthe nifD region were deleted; however, the nifH promotor and thenifH ribosome-binding site were unaltered. The deleted nif regionwas replaced by a Mst Il-Apa I DNA fragment that includes thefdxAcoding region but does not include fdxA transcription initiationsignals. The dot in the fdxA gene represents the location of the Cysto Ala codon substitution. bp, Base pairs.

raphy by using essentially the same conditions as for thenative protein (15), except that all procedures were carriedout anaerobically. Crystals were mounted under anaerobicconditions in 1.0-mm sealed glass capillaries by using asynthetic mother liquor of 0.1 M Tris HC1 (pH 8.0) in 5.1 M(NH4)2SO4. Crystals of native FdI are tetragonal, spacegroup P41212, with a = b = 55.2 A, c = 95.2 A, and onemolecule per asymmetric unit (5). Precession photographs ofthe C20A crystals showed the same systematic absences asfor native crystals but distinct differences in the intensities to3.0-A resolution and unit-cell dimensions of a = b = 55.0 Aand c = 95.4 A. A single crystal of dimensions 0.5 x 0.5 x1.5 mm was used for data collection. Data were collected asfor the native FdI (5). Data were indexed, integrated, re-duced, merged, and scaled (16). The 1.9-A resolution data hasRsymm on IF! of9.5% in point group 4/mmm and 8.3% in pointgroup 422 for all 55,860 observations with Fl > 0.0 of 11,697possible reflections [average I/o-(I) 16.1].

The C20A FdI data to 2.3 A were scaled against the nativedata in shells of sin O/A by using an anisotropic shape factor(Rmerge on IFI of 30.0%). A difference Fourier map withcoefficients IFnativel - IFC20AI and phases calculated fromthe native structure refined at 1.9-A resolution (5) hadthree significant features with respect to the native structure:a positive peak of +250 at the S-y position of Cys-20, a pair ofpeaks +200/-200 on either side of SY of Cys-24, indicatinga shift toward Fe4 of the [4Fe-4S] cluster, and a number ofpeaks of +100/-100 associated with the [Fe-S] clusters,suggesting a concerted shift of the entire molecule due to thechange in origin of the unit cell of0.35 A. The model with Fe4and SY ofCys-20 omitted was refined against the 2.3-A mutantdata with the program X-PLOR (17), decreasing the R-factorfrom 0.40 to 0.27. The resulting phases were used to calculatetwo maps with the mutant data: a (21F01 - lFCI) map, whichconfirmed the presence of a [4Fe-4S] cluster with SY ofCys-24 as the ligand to Fe4, and a Bijvoet difference Fouriermap (18), which independently confirmed the presence offour Fe atoms in this cluster.A (21Fj1 - jFCj) Fourier map to 1.9-A resolution was

calculated using the partially refined model with residues20-24 omitted. These residues and the [4Fe-4S] cluster werefit to the mutant electron density. The model was refined toR of 0.279 for all 9792 reflections with IFI > 0.0 in the range8.0-1.9 A. Refinement of individual isotropic temperaturefactors and inclusion of 31 waters with B - 40 A2 reduced Rto 0.232. The final model has rms deviations from ideality of0.019 A for bonds and 3.67° for angles.For comparison of the structures (Fig. 2), coordinates for

the native structure were taken from the 1.9-A resolutionrefinement (5). The structures were superimposed by leastsquares minimization of all pairs of backbone N, Ca, and Catoms, except those in residues 19-25 and residues 38-46

a

b

FIG. 2. Coordinates of native andC20A Fdls from 1.9-A resolution refine-ments of the structures. The structures

-39 were aligned by least squares fit of back-bone N, Ca, and C atoms of residues1-18, residues 26-37, and residues 47-

45 106. Heavy lines, C20A FdI; light lines,native FdI. The [4Fe-4S](Sy)4 clusters areshown with residues 19-25 and residues38-46 (a) and residues 20-24 (b).

Biochemistry: Martin et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

(Fig. 2a). The rms deviation is 0.22 A. For residues 19-25 andresidues 38-46, the rms deviation is 0.27 A. The 0,)4 torsionangles of the two structures deviate by less than ±15°everywhere except at residues 22-24 and 46 (Fig. 2b). Thealtered torsion angles remain within allowed regions of theRamachandran plot.

Spectroscopy. EPR spectra were obtained using a BrukerESR-200D, interfaced with an Oxford ESR-9 flow cryostat.Absorption and circular dichroism (CD) spectra were ob-tained using a Cary 17 and a Jasco J-500C (7, 19).C20A FdI was examined in the Na2S204-reduced and

02-oxidized states. Anaerobically obtained triclinic crystalsofC20A FdI were redissolved in degassed 100 mM potassiumphosphate (pH 7.4) containing 2 mM Na2S204. After anaer-obic spectroscopy, the protein was allowed to air-oxidize.Oxidation was monitored by EPR and was repeated until noEPR increase was seen (z30 min thawed time). After spec-troscopy Na2S204 was added to a concentration of 2 mM.After a 30-min incubation, spectra were identical to thosemeasured initially. The extinction coefficient of oxidizedC20A FdI has been assumed to be identical to that of nativeFdI, E4W = 29,800.

RESULTS AND DISCUSSIONProteins that contain [4Fe-4S] clusters can often be recog-nized by the presence of a -Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-sequence, which supplies three of the four cluster ligands (1,20). The fourth ligand is supplied by a single cysteine residuefrom some remote portion of the protein that is frequentlypart of a -Cys-Pro-Val- sequence (20). As a result of thisarrangement of ligands, [4Fe-4S] clusters generally bridgetwo portions of a protein. For AvFdI the [4Fe-4S] clusterbridges two lengths of polypeptide with cysteine residues 39,

a

b

42, and 45, supplying three ligands and the remote -Cys-Pro-Val- sequence (residues 20-22) providing the fourthligand (3-5). The site-directed mutation ofCys-20 to Ala was,therefore, expected to provide information concerning theextent to which the "bridging" cluster influences proteinfolding, in addition to information concerning what type ofcluster can be formed in the absence of the remote cysteineligand. Our results show that replacement of Cys-20 by Alaleads to the formation of a new [4Fe-4S] cluster in which theligating function of Cys-20 is replaced by Cys-24.

Structure of C20A FdI. The C20A site-directed mutant ofAvFdI was constructed in vitro, expressed in A. vinelandiiunder control of the nifI promoter, anaerobically purified,crystallized, and examined by x-ray crystallography. Fig. 2shows a superposition of the native and C2OA FdI structuresin the region of the [4Fe-4S] cluster and at the site ofmutation, residues 20-24. The remainder of the protein,including the [3Fe-4S] cluster, was essentially unaffectedeither by the mutation or by the anaerobic treatment of theprotein. These data confirm the conclusion from previousstudies (8) that the [3Fe-4S] cluster ofAvFdI is not simply anartifact of oxygen degradation.Comparison ofthe native and C20A structures in the region

ofthe [4Fe-4S] cluster (Fig. 2) demonstrates that the mutationof Cys-20 to Ala has caused the structure of the protein torearrange such that Cys-24, which is a free cysteine in van derWaals contact with the [4Fe-4S] cluster in the native protein,becomes the fourth ligand to a [4Fe-4S] cluster in the C20Amutant FdI. This is further illustrated in Fig. 3, which showsthe 1.9-A resolution (21F01 - IFcI) electron density map for theC20A FdI at the Cys-20 to Ala mutation and at the new Cys-24to Fe bond.There are three components to the structural rearrange-

ment illustrated in Figs. 2 and 3. (i) The new 0, fi torsion

FIG. 3. Electron density map forC20A FdI calculated with coefficients(21F01 - IFJI) and 9792 reflections in theresolution range 8.0-1.9 A. (a) Residues16-23 in the sequence Cys-Val-Glu-Val-Ala-Pro-Val-Asp: the side chains ofVal-17 and Val-19 are at the lower rightand left, respectively; Ala-20 is at thecenter. Contoured at 0.10 of the maxi-mum density. (b) Residues 21-26 in thesequence Pro-Val-Asp-Cys-Phe-Tyr andFe4 of the [4Fe-4S] cluster: Pro-21 is atthe top and Tyr-26 is at the lower left. TheAsp-23 side chain is viewed edge-on invery weak density. Contoured at 0.10 and0.30 of the maximum density.

600 Biochemistry: Martin et al.

Proc. Natl. Acad. Sci. USA 87 (1990) 601

transFe

9+ 900- -

C0,00

FIG. 4. Plot of Q,-C-Spy-Fe torsion angles viewed down the CPto SY bond. The angles for 30 cysteines in five proteins and eight[Fe-S] clusters are plotted. The proteins are native and C20A 7Feferredoxin, 8Fe ferredoxin (21), HiPIP (22), and 2Fe ferredoxin (23).The four angles outside the preferred g+ or g- range occur at Cys-24of C20A 7Fe ferredoxin (1660), Cys-63 of HiPIP (126°), Cys-46 ofHiPIP (2000), and Cys-49 of 2Fe ferredoxin (2020).

angles at residues 22-24 have the effect of rotating the Cys-24Ca-Cp6 bond approximately 450 and displacing it approxi-mately 1 A toward the cluster (Fig. 2b). As a result, S. ofCys-24 moves 2.0 A. (ii) There is a smaller but concertedreorientation of the chain containing the Cys-39, Cys-42, andCys-45 ligands that rotates the cluster by approximately 100;this allows the fourth coordination site of Fe4 to be directedtoward the SY of Cys-24 (Fig. 2a). (iii) The side chain ofCys-24 adopts an extended (trans) conformation allowing theCaX-Fe4 distance to increase from 3.7 A in the native proteinto 4.6 A in the mutant. With regard to the first component ofthe rearrangement a large shift in the position of Asp-23 andits sidechain (Fig. 2b) is permissible because this residue is onthe surface of the molecule and extends into solvent in bothcrystals. With respect to the second component, a concertedreorientation of the triplet of cysteines in residues 39-45 isaccommodated by 4, 4i torsion angle changes of Glu-46; thisside chain is also on the surface. (iv) The C,-Cs-Sg-Fe torsionangles of cysteine ligands in [Fe-S] proteins are normally g+or g- (Fig. 4). However, the angle at Cys-24 in the mutant

0

xU.;

70

60 __\40 _ \

30

20

iO-10

300 350 400 450 500 550 600 650

A, nm

protein is trans, a rarer but allowed conformation observed inChromatium vinosum high-potential iron protein (HiPIP) (22)and Spirulina platensis [2Fe-2S] ferredoxin (23).The rearrangement of the structure in the C20A protein

leads to several new interactions. Compared to the nativeprotein, six hydrogen bonds are lost, three are gained, andseven involving residues 18-25 and residues 38-46 are pre-served (5). The new position of the Asp-23 side chain inproximity to the Glu-38 side chain (Fig. 2a) is apparently thecause for its becoming disordered; the density for this residueis very weak (Fig. 3b). Overall the mutant and native struc-tures have similar B values. The methyl group of Ala-20 is invan der Waals contact with Sy of Cys-24. The [Fe-S] clustersin the C20A and native (5) structures have similar geometry.The same constraints were used in both refinements. The[4Fe-4S] cores of the two structures superpose to within 0.08A. By comparison, the [3Fe-4S] clusters ofthe two structuresagree to within 0.05 A. SY-Fe-S angles in the two structuresdo not vary outside the normal range (24). At 1.9-A resolutionit appears that the [4Fe-4S](Sy)4 cluster moves essentially asa rigid body. In addition, five possible NH ... S hydrogenbonds in the native protein are preserved and no newNH ... S bonds are gained. Also, the pattern of Ca-Cp-SY-Fetorsion angles for Cys-39, Cys-42, and Cys-45 remains g ,g, and g- (Fig. 4). The Cys-Cys-Cys moiety (residues 39, 42,and 45, respectively) moves as a stereochemically restrainedgrouping hinged at Glu-46; the 47 atoms of residues 39-45 canbe aligned to within 0.17 A. The trans conformation atCys-Phe (residues 24 and 25, respectively) in the mutantmarks a real difference at the [4Fe-4S](Sy)4 cluster due tomutation.The data presented here demonstrate that, when the AvFdI

[4Fe-4S] cluster is denied one of its normal cysteine ligands,it is able to use another cysteine that is nearby. A similarrearrangement may have occurred after site-directed muta-genesis of the [4Fe-4S] cluster containing Bacillus subtilisglutamine phosphoribosylpyrophosphate amidotransferase(25) and after mutation of two conserved cysteine residues(nifD, position 183, and nifK, position 153) to serines in theA. vinelandii nitrogenase molybdenum-iron protein (26).Thus, in both cases alteration of putative cluster ligandsresulted in the production of active enzyme (25, 26).

Stability and Redox Properties. There are two features ofthe [4Fe-4S] cluster of the C20A protein that might beexpected to affect its stability, redox properties, or both

X, nm

FIG. 5. Absorption (A and C) and CD (B and D) of native Fdl (solid line) and C20A FdI (dashed line) in 100 mM potassium phosphate (pH7.4). (A and B) 02-oxidized state. (C and D) In 2 mM Na2S204.

c

I

Biochemistry: Martfn et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

4- g

50 G* 10

H No

FIG. 6. EPR of 02-oxidized native FdI (a) and C20A FdI (b) at10 K. Native FdI was 41.4 ,uM and C20A FdI was 7.3 ,uM in 100 mMpotassium phosphate (pH 7.4). Microwave power, microwave fre-quency, and modulation amplitude were 1 mW, 9.59 GHz, and 5 G,respectively. The gain was 2 x 104 and 1.25 x 105 for native FdI andC20A FdI, respectively.

based on comparison of the sequences of other ferredoxins.(i) The [4Fe-4S] cluster of the C20A protein no longerreceives its remote cysteine ligand from the Cys-Pro se-

quence, which is highly conserved in bacterial ferredoxins(27). (ii) There is no longer a free cysteine residue in thevicinity of the [4Fe-4S] cluster of the C20A protein. Such afree cysteine is found in all 7Fe ferredoxins but is missing inthe smaller 8Fe ferredoxin from Clostridium pasteurianum(20, 27).EPR and near-UV/visible absorption and CD spectra were

obtained for the Na2S204 reduced and 02-oxidized states ofC20A FdI. The absorption spectrum of C20A FdI is verysimilar to that of native FdI in both oxidation levels (Fig. 5 Aand C). The complete reversibility of the changes demon-strates that, like native FdI, C20A FdI is 02-stable.The CD spectra of [Fe-S] proteins show substantially more

structure than the corresponding absorption spectra. Sincethe CD of achiral [Fe-S] clusters originates in their chiralprotein environment, CD generally varies significantly withprotein (19). The CD of C20A FdI differs substantially fromthat of native FdI in both oxidation levels (Fig. 5 B and D).This large change in CD is consistent with significant re-arrangement of the environment of the [4Fe-4S] cluster andis presumably due to changes in protein 0, 4i torsion anglesand to the trans conformation of the Cys-24 ligand versus thenormal g+ for Cys-20.The EPR of oxidized C20A FdI was found to be very

similar in g-value, shape, temperature dependence, and mi-crowave power dependence to that of oxidized native FdI(Fig. 6). Since the EPR is attributable to the [3Fe-4S]+1cluster (7), this cluster must be essentially identical in struc-ture in both ferredoxins. No other EPR was observed from g= 1 to 14, and integration of the C20A FdI spectrum usingnative FdI as a standard yielded 0.95 spin per molecule.Dithionite at pH 7.4 reduces the g =2.02 EPR to -5% of theoxidized intensity in both ferredoxins; no new EPR signalsappear from g = 1 to 14.The spectra of C20A FdI are fully consistent with the

crystallographic data showing one [4Fe-4S] and one [3Fe-4S]cluster, with considerable rearrangement of protein. Thelatter cluster is shown to be [3Fe-4S]+' and [3Fe-4S]° in thepresence of 02 and dithionite, respectively, whereas the

former is [4Fe-4S]+2 under both conditions. Although theexact reduction potential of the [4Fe-4S]+211' pair in C20AFdI is unknown, the data demonstrate that it is still inacces-sible to reduction by dithionite at pH 7.4.

CONCLUSIONWe have applied site-directed mutagenesis to the study of acrystallographically characterized [Fe-S] protein. The resultsshow that when a [4Fe-4S] cluster is denied one of its normalcysteine ligands it can rearrange the protein to obtain a newcysteine ligand.

We are indebted to A. H. Robbins for helpful discussions and G. P.Gippert for graphics. This research was supported by a NationalScience Foundation grant (DMB-8718470) to B.K.B., a NationalScience Foundation grant (DMB-8706460) to P.J.S., and a NationalInstitutes of Health grant (GM36325) to C.D.S.

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