site-directed mutagenesis of azotobacter vinelandii ferredoxin i

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry ' and Moleculat ' Biology, Inc

Vol. 266. No. 32. Issue of November 15, pp. 21563-21571,1991 Printed in U. S. A.

Site-directed Mutagenesis of Azotobacter vinelandii Ferredoxin I CHANGES IN [4Fe-4S] CLUSTER REDUCTION POTENTIAL AND REACTIVITY*

(Received for publication, June 6, 1991)

Siiri E. Iismaa$$, Ana E. Vhquez$lI, Gerard M. Jensenll, Philip J. Stephens//, Julea N. Butt**, Fraser A. Armstrong**, and Barbara K. Burgess$ $$ From the Departments of $Molecular Biology and Biochemistry and **Chemistry, University of California, Irvine, California 9271 7 and the 1) Department of Chemistry, University of Southern California, Los Angeles, California 90089-0482

We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rear- ranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectro- scopic results show that all three proteins exhibit the same general redox behavior during 02-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a -150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)z- to form a paramagnetic species previously proposed to be a cysteinyldisulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.

Iron-sulfur ([Fe-SI) proteins are ubiquitous in Nature, where they carry out both redox and non-redox functions (1). Given the importance of this class of proteins and the wide diversity of protein-bound [Fe-S] cluster types, redox properties and reactivity, it is not surprising that they have been the subject of extensive investigation. Recently, we have begun to study the relationships between protein primary structure and [Fe-SI cluster structure and reactivity via site-

DMB-8718470 and DMB-8706460 and by National Institutes of * This work was supported by National Science Foundation Grant

Health Grant R01-GM45209. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: The Heart Research Institute 145-147 Missen- den Rd., Camperdown, N.S.W., 2050 Australia.

ll Supported in part by a National Institutes of Health Pre-doctoral Training Grant.

$$ To whom correspondence should be addressed: Dept. of Molec- ular Biology and Biochemistry, University of California, Irvine, CA 92717.

directed mutagenesis of Azotobacter vinelundii ferredoxin I (AvFdI) (2). AvFdI was chosen for this study because it is a small protein (polypeptide M, - 12,700) that has been characterized by both x-ray crystallography (3-5) and spectroscopic methods (6, 7 and references therein). It contains two different types of [Fe-SI cluster: one [3Fe-4S]+/O cluster and one [4Fe-4S]2+'" cluster.

Sequence comparisons (Fig. 1) show that AuFdI is a member of a closely related class of ferredoxins found in aerobic organisms, all of which are believed to contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. As shown in Fig. 1, in these (and many other) proteins the presence of a [4Fe-4S] cluster can be recognized by a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence which supplies three of the four required cluster ligands (8,9). The fourth cysteine ligand to the [4Fe-4S] cluster generally comes from some distal region of the polypeptide sequence which results in the cluster bridging two portions of the protein (9). Recently, we reported the structure of a variant of AvFdI (designated C20A) in which the remote cysteine ligand to the [4Fe-4S] cluster (C20) was replaced by an alanine (2). The results showed that when the [4Fe-4S] cluster was denied one of its normal cysteine ligands, a [4Fe- 4S] cluster was nevertheless assembled. However, rearrangement of the protein was required to provide a new cysteine ligand.

The replacement ligand in the C20A protein is cysteine 24. In the native protein this cysteine is in van der Waals contact with the [4Fe-4S] cluster (5). Although its influence on the native [4Fe-4S] cluster is unknown, Fig. 1 shows that C24 is conserved in this class of 7Fe ferredoxins. To investigate the role of this residue further, we have constructed an additional variant of AvFdI by site-directed mutagenesis in which the cysteine a t position 24 is converted to an alanine. The structure of this protein (designated C24A) is described in the accompanying paper (10). Here we compare the redox properties, chemical reactivity, and function of the native, C20A, and C24A proteins. This study allows us to address the role of these cysteines in a commonly occurring [Fe-SI protein sequence and structural motif.

EXPERIMENTAL PROCEDURES

Materials-Native and mutant variants of AvFdI were purified and crystallized as described previously (4, 7) except that, as a precau- tionary measure, the C20A and C24A proteins were initially purified anaerobically in the presence of dithionite. Polyclonal antibodies were raised against native FdI in New Zealand White rabbits at Bethyl Laboratories, Montgomery, TX. The IgG fraction of the sera was purified with ammonium sulfate precipitation using a published procedure (11). Goat anti-rabbit IgG conjugated to horseradish per- oxidase was obtained from Cooper Biomedical, Inc., Malvern, PA. Ampholines for two-dimensional gel electrophoresis were from LKB, Bromma, Sweden. Acrylamide and sodium dodecyl sulfate were from

21563

21564 Site-directed Mutagenesis: AvFdI

1- [4Fe - 4S] -1 I

10 FIG. 1. Sequence comparison of C K Y T D C V E V C P V D C F Y E G P N F L V I H P D E C I D C A L C E P E C P

20 24 30 I40 I I 50

A. uinela&ii ferredoxin 1 to other C K Y T D C V E V C P V D C F Y E G P N F L V I H P D E C I D C A L C E P E C P C K Y T D C V E V C P V D C F Y E G P N F L V I H P D E C I D C

7Fe ferredoxins. Identical residues to C K T D C V E V C P V D C

Key: Au, A. uinelandii ferredoxin I (35); the A. uinelandii sequence are boxed.

Po,-Pseudomonas ovalis ferredoxin I (36); I I Ps, Pseudomonas stutzeri ferredoxin (37); Rc, Rhodobacter capsulatus ferredoxin I1 (38); Sg, Streptomyces griseus ferredoxin (39); Ms, Mycobacterium smegmatis ferredoxin (40); Tt, Thermus

60 70 100

thermophilus ferredoxin (41).

Bio-Rad. Tris, MES,' HEPES, TAPS, glycine, dithiothreitol, kana- mycin, neomycin, and tobramycin were obtained from Sigma. Restric- tion enzymes, T4 DNA ligase, T4 polynucleotide kinase, SI nuclease, the Klenow fragment of DNA polymerase I, bacteriophage M13mp18, and plasmid pUC9 were purchased from Bethesda Research Labora- tories and used as recommended by the suppliers. Plasmid pKT230 was obtained from K. N. Timmis, Department of Microbiology, Technical University, Braunschweig, Germany (12). [y3*P]ATP (-3000 Ci/mmol) and [cP~'P]~ATP (-3000 Ci/mmol) were obtained from Amersham Corp. The M13 universal sequence primer and exonuclease I11 were purchased from New England Biolabs, Beverly, MA. K3Fe(CN)s (AR grade) was from Mallinckrodt.

Mutagenesis of f d d and Expression of the Altered FdZs-Oligonu- cleotide-directed in uitro mutagenesis was performed using an Amer- sham Corp. oligo-directed in uitro mutagenesis kit based on the method of Eckstein and co-workers (13, 14). The template for mutagenesis was a 2.6 kilobase S m I fragment derived from pMLl (15) carrying the wild type jdxA gene cloned into M13mp18 such that the direction of fdxA transcription is toward the EcoRI site of M13mp18. The oligonucleotide used for construction of C2OAFdI was described previously (2). The oligonucleotide used for C24A mutagenesis has the sequence TGCCCGGTAGACW TTTCTACGAACGGCCG. The underlined nucleotides represent substitutions that change codon 25 within the fdxA gene from TGT to GCT, resulting in the substitution of alanine for cysteine at position 24 in the FdI protein sequence. The success of the mutagenesis was confirmed at the DNA level by dideoxy DNA sequencing and at the protein level by x-ray crystallography (10).

The genotypes of all A. uinelandii strains used in this study are shown in Fig. 2. Three different expression systems were used to produce the C20A protein in A. uinelandii. Expression system I, which involved replacing the chromosomal nifH gene with the CZOAfdxA gene, is described elsewhere (2). Expression system I1 involved construction of an A. uinelandii strain, designated JG2OA (Fig. 2) that is identical to the wild type except that the native fdxA gene, in its normal location on the chromosome, has been replaced by the C20A version of fdxA. The methodology used in the construction of that strain was previously described (16). Expression system I11 involved subcloning the 2.6 kilobase SmaI fragment derived from pMLl (15), carrying the C20A version of the fdxA gene, into the broad host range multicopy plasmid pKT230 (12). This plasmid was constructed and maintained in Escherichia coli strain C600 and introduced into A. uinelandii via a triparental mating with E. coli strain HBlOl/ pRK2013 which carries the helper plasmid pRK2013 (17). When this system was used to express native fdxA (strain JG100/pDB214 in Fig. 2) it resulted in a -50-fold overproduction of AuFdI. The overex- pressed native protein was shown to be identical to that purified from wild type cells by x-ray crystallography and spectroscopic characterization? The C24A version of fdxA was expressed using systems I1 and 111. For physiological studies two additional strains JG103 and JG118 (Fig. 2) were constructed using previously described methodology (16).

Mutant Analysis-Unless otherwise indicated, cell growth, extract

' The abbreviations used are: MES, 2-(N-morpholino)- ethanesulfonic acid; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; TAPS, N-tris-(hydroxymethyl)methyl-3-aminopro- panesulfonic acid.

S. E. Iismaa, A. E. Vazquez, G. M. Jensen, P. J. Stephens, J. N. Butt, F. A. Armstrong, and B. K. Burgess, unpublished results.

preparation, two-dimensional gel electrophoresis, and Western analysis were performed as described previously (16).

Spectroscopy-Spectroscopic studies of C20A, C24A, and native FdI were carried out using methods previously described (2, 7). EPR spectra were obtained using a Bruker ER-2OOD spectrometer, interfaced with an Oxford Instruments ESR-900 flow cryostat. Integra- tions of EPR spectra were carried out using native FdI as a standard. Absorption spectra were obtained using a Cary 17 spectrometer, and CD spectra were obtained using a Jasco J-500C spectropolarimeter. Absorption and CD measurements were carried out using small volume cylindrical cells with fused quartz windows (Optical Cell Co.). For anaerobic measurements, the cells were loaded in an 02-free (0, < 1 ppm) glove box (Vacuum Atmospheres, Hawthorne, CA) into an O-ring sealed holder with fused quartz windows. The extinction coefficients of C20A and C24A were taken to be the same as that of native FdI = 29,800 M" cm").

Voltammetry of Native, C20A, and C24A FdI-Solutions of FdIs were dialyzed anaerobically in an ultrafiltration cell (Amicon 8MC fitted with microvolume assembly and YM-5 membrane) against a mixed buffer electrolyte (5 mM in each of acetate, MES, HEPES, and TAPS with 0.1 M NaCl) adjusted to the desired pH (range 5.5-8.5) at 3 "c. Final protein concentrations were approximately 100 p M as judged from the absorbance at 400 nm. All reagents were of the highest grade commercially available. Water used for preparing solutions was prepurified using a Barnstead "Nanopure" deionizer.

For the greater part of this study, we employed square-wave voltammetry because of its greater sensitivity and resolution, but several complementary measurements were made with cyclic voltammetry. Square-wave voltammetry was carried out using a Princeton Applied Research model 273 electrochemical analyzer interfaced to an AT 286-12 computer. DC cyclic voltammetry was carried out using an Ursar Instruments potentiostat. The electrochemical cell was similar to that described in an earlier publication (18). The edge-oriented pyrolytic graphite working electrode and a platinum gauze auxiliary electrode were positioned in the sample compartment (capacity 0.5 ml). This was maintained at 3 "C by immersion in a circulation bath. The sample solution was made anaerobic by passing a current of purified Ar over the surface. The saturated calomel reference electrode was located in a thermostatted side arm (25 "C) linked to the sample compartment via a Luggin capillary tip. For square-wave voltammetry, a 1.0 pF capacitator was placed between the auxiliary electrode and the reference input of the differential electrometer. All potentials were adjusted to the standard hydrogen electrode scale by adding 244 mV. Prior to each voltammetric scan, the pyrolytic graphite working electrode surface was polished with an aqueous slurry of 1.0-pm A1203 (Buehler micropolish) and then sonicated thoroughly. In each set of experiments, the voltammetric response was induced as described previously (18) by stepwise additions of small aliquots of neomycin or tobramycin from 0.2 M, pH 7.0, stock solutions.

K+'e(CN)$ Oxidation Experiments-Unless otherwise indicated all experlments were carried out anaerobically in 0.1 M potassium phosphate buffer, pH 7.5. Concentrations of stock solutions of K,Fe(CN)6 were confirmed, after dilution, by absorption spectroscopy [e420 = 10201 (19). All solutions of K3Fe(CNI6 were freshly prepared to minimize hydrolysis. Reaction with Fe(CN)i- was monitored via absorption spectroscopy. CD and EPR spectra were measured subsequent to equilibration.

RESULTS AND DISCUSSION

We now have available the x-ray structures of three variants of FdI, the native protein (3-5) and the C20A (2) and C24A

Site-directed Mutagenesis: AvFdI 21565

&Strain Chromosome Plasmid Nitrogenase __ Fd I Fld

p nif H D K fdxA nif F W T U-C3 -

pC2OAfdxAnifK &u p nit H D K CZOAfdxA nif F

C20A -

JG20A -

p nit H D K CZOAfdxA IoCZfuSlon JG103 -

p nit H D K C24AfdxA nit F JG106 -I -

JGIOO/pAVIOO p nit H D K Kan a"'? & pKT230IC24AfdxA

p nit H D K C24AfdxA IocZfusion

J G l l 8 -I I) -

JGIOO/pDB214 p nit H D K Z A t d x ?

pKT230IfdxA

FIG. 2. Genotypes of all A. vinelandii strains used in this study. Key: nifHDK encodes the Fe protein polypeptide and the a- and &subunits of the MoFe protein, respectively; fdxA encodes the AuFdI polypeptide; nifF encodes A. vinelandii flavodoxin (Fld). All strains were constructed as described under "Experimental Proce- dures."

(10) site-directed mutants. The results show that all three forms contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structural environment immediately surrounding the [3Fe-4S] cluster in all three proteins is very similar, except that the entire C24A protein exhibits larger B-factors than the other two proteins (10). The structural environment surrounding the [4Fe-4S] cluster in the native and C24A proteins is extremely similar, except for a pronounced difference in B- factor, and, of course, the notable absence of the sulfur atom at position 24. The [4Fe-4S] cluster environment in C20A is also missing a free cysteine, but in that case there has addi- tionally been a large structural rearrangement in that region of the protein.

O2 Oxidation and Dithwnite Reduction-Unlike many [Fe- S] proteins, native FdI is stable toward 0 2 . In the 02-oxidized state its [3Fe-4S] and [4Fe-4S] clusters exist in the 1+ and 2+ oxidation levels, respectively (7 and references therein). Near neutral pH, dithionite reduces the [3Fe-4S]+ cluster nearly completely to the zero oxidation level, but does not reduce the [4Fe-4SI2+ cluster (7). The latter cluster can, however, be partially reduced to the 1+ level by dithionite at higher pH (20). The reduction potential of the [3Fe-4S] cluster in native FdI is approximately -420 mV (uersw standard hydrogen electrode) (21) while that of the [4Fe-4S] cluster is much lower (20). Cyclic voltammetric studies of native Azotobacter chroococcum ferredoxin I (AcFdI) have found reduction potentials of its analogous clusters to be approximately -420 and -640 mV (18). The potential of the AcFdI [3Fe-4S] cluster is reported to exhibit a substantial pH dependence (18), which appears to originate in protonation when the cluster is in the reduced, zero oxidation level (7,22).

Recently, we have reported the visible-UV absorption and CD of the 02-oxidized and dithionite-reduced, pH 7.4, states of the C20A variant of FdI (2). Like native FdI, C20A is O2 stable and near neutral pH its [3Fe-4S] and [4Fe-4S] clusters are, respectively, reducible and nonreducible by dithionite. In this work we have examined C24A in a similar manner, C24A was purified anaerobically in the presence of dithionite, Spec- troscopic study of this protein, after air oxidation and after subsequent rereduction by dithionite, shows that C24A is stable to 02 and that its dithionite reduction behavior is also

parallel to that of native FdI. The visible-UV absorption and CD spectra of 02-oxidized and dithionite-reduced C24A are shown in Fig. 3 and 4, with the analogous spectra of native FdI. The spectra of C24A and native FdI are extremely similar. While this is also true for C20A in the case of visible- UV absorption, the CD spectra of C20A and native FdI differ substantially (2). The much greater similarity of the CD of C24A and native FdI shows that the environment of the [Fe- S] clusters is much less changed in C24A than in C20A, a conclusion in agreement with the results of x-ray crystallography (2, 10). The near identity of the absorption and CD spectra of C24A and native FdI also strongly supports the interpretation of the C24A/native FdI difference Fourier map discussed in the accompanying paper. That map shows four strong peaks of varying magnitude, associated with the Fe atoms of the [4Fe-4S] cluster. These are interpreted as being due to inefficient packing in the vicinity of the C24A [4Fe- 4S] cluster giving rise to a higher B-factor for that cluster. If those peaks had been due to lower iron occupancy in the [4Fe- 4S] cluster site of the C24A protein, the optical spectra of the C24A protein would have been substantially different from that of the native protein. As shown in Figs. 3 and 4, this is not the case.

The EPR of oxidized C24A was also found to be essentially identical in g value, shape, and temperature dependence to that of the oxidized native FdI (Fig. 5), as was the EPR of oxidized C20A (2). This EPR arises from the [3Fe-4S]+ cluster

250 350 450 550 850

h ( n d

FIG. 3. Absorption spectra of native FdI (a and c) and C24A-FdI ( b and d ) in 100 mM potassium phosphate buffer, pH 7.4. Spectra a and b were in the 02-oxidized state. Spectra c and d were in - mM Na2S204. FdI concentrations were -40 p ~ .

20-

15 - B A<

10

5

0

-5

I l l l l l / / 1 1

FIG. 4. Circular dichroism spectra of native FdI (a) and C24A-FdI (b) in 100 mM potassium phosphate buffer, pH 7.4. The samples in A were in the O,-oxidized state, while those in B were in -2 mM Na2S204. Protein concentrations were -40 pM.

300 350 400 450 5 0 0 550 600 650 I("rn1


FIG. 5. EPR spectra of 02-oxidized C24A-FdI (a) and native FdI (b ) at -10 K. C24A-FdI was 74.1 PM and native FdI was 41.4 PM in 100 mM potassium phosphate buffer, pH 7.4. Microwave power, microwave frequency, modulation amplitude, and gain were 1 mW, 9.59 GHz, 5 G, and 1.6 X lo4, respectively.

(7,23) confirming that its environment is essentially identical in all three proteins. As with the oxidized native and C20A proteins, no other EPR was observed from g = 1-14. Dithio- nite at pH 7.4 reduces the g -2.01 EPR to -5% of its oxidized intensity for all three proteins. No new EPR signals appear from g = 1-14 upon dithionite reduction of C24A at pH 7.4, demonstrating that like the native (20) and C20A (2) proteins, the C24A [4Fe-4S] cluster must have a very low reduction potential.

Electrochemical Characterization of Native, CZOA, and C24A"In the absence of aminocyclitol promoters, the voltammetric responses of native, C20A, and C24A FdIs were poor and ill-defined. By contrast, well-defined waves due to protein redox couples were revealed upon introducing neomycin or tobramycin to final concentrations of -2 mM. Be- cause of the known pH dependence of the [3Fe-4S]'/O cluster potential for the native protein (18), experiments were carried out over the pH range 5.5-8.5. Cyclic voltammograms of the native protein were very similar to those reported earlier for the analogous FdI from A. chroococcum measured under identical experimental conditions (18). This provides further sup- port for the suggestion made in that paper, that AcFdI and AvFdI have essentially identical cluster environments.

Square-wave voltammograms directly comparing the native, C20A, and C24A proteins at pH 7.8 are shown in Fig. 6. Two prominent resultant current maxima (sum of forward and reverse-pulse currents) are observed for all proteins. These are termed couples (peaks) A and B in order of decreas- ing potential. A third redox couple (C) having a lower intensity is observed as a peak for C24A, as a shoulder on peak B for the native protein, and as a slight broadening of peak B for C20A.

[3Fe-4S]'/" Cluster Potentials-By analogy with an earlier report on FdI from A. chroococcum, peaks A of Fig. 6 are assigned to the one-electron reduction and reoxidation of the [3Fe-4S]+ cluster (18). For native AvFdI the potential for this couple is approximately -425 mV at pH 7.8. Examination of the data for peaks A in Fig. 6 clearly shows that the reduction potential for the [ 3Fe-4S]+Io couple is unaffected in either the C20A or the C24A variants. Plots of reduction potentials for all three proteins as a function of pH are given in Fig. 7 where it is clear that the data for the [3Fe-4S]+/O couple are virtually superimposable. This result is fully consistent with the lack of structural change in that region of the protein observed by x-ray crystallography (2, 10).

Native

C2OR ~

I . I , I , I

-1000 -800 -600 -400 - 2 0 0

E / mU U S SHE

FIG. 6. Square-wave voltammograms at PGE electrodes of solutions of AvFdI, native, and mutant forms. Shown are resultant peak currents for scans made in the reductive direction. Protein concentrations are 100 WM in 0.1 M NaC1, pH 7.8, mixed buffer (5 mM each in acetate, MES, HEPES, and TAPS), containing 2 mM neomycin. Temperature is 3" c. Square-wave frequency is 60 Hz, pulse amplitude 40 mV, and the potential step increment is 1 mV. Under more alkaline conditions, i.e. typically pH > 7.8, we found that all potentials measured from square-wave resultant peak maxima became slightly more positive (-5-10 mV) upon increasing the neomycin or tobramycin concentrations from 0.5 to 2.0 mM. At pH values below pH 6.6, neomycin was found to cause protein precipitation; thus tobramycin was used instead, without causing any noticeable discontinuity in results. With voltammograms of such complexity, we did not find it meaningful to apply rigorous tests for diffusion control. This would normally require peak currents to be proportional to the square root of the frequency (24). We found that the relative amplitudes of the square-wave voltammetric responses for each couple in individual runs were somewhat dependent upon square-wave frequency, pulse height, and the time that the electrode was in contact with the solution. However, we observed that resultant peak potentials for couples A and B were essentially invariant (within a margin of 5 mV) with frequency, over at least the range 15-80 Hz. From this we could conclude that our results are neither sensitive to kinetic effects nor altered by the increasing contribution from strongly ad- sorbed molecules expected at higher frequency. Resultant peak potentials for A or B measured by scanning in the oxidative direction, following prescan potential incubation at -950 mV versus standard hydrogen electrode, were within 2 mV (more positive) of those measured for the reductive direction. The peak amplitude for couple C, as measured relative to amplitudes of A and B, showed greater sensitivity to pulse height. Furthermore, when scanning in the oxidative direction, the response for C merged into B and the peak position was not measurable in most cases. Due to this obvious kinetic complexity, effective E"' values for C are not determinable with any certainty from these experiments. Thus, in considering peak C we chose to limit our analysis to a comparison of peak potential values for reductive scans at a frequency of 30 Hz. These were clearly defined for the C24A mutant protein above pH 6.6, but for the native form data were limited to pH 7.8 and above.

Examination of native AvFdI and native AcFdI by CD and magnetic circular dichroism spectroscopy has previously shown that the one-electron reduced [3Fe-4SIo cluster exists in two forms depending upon the pH (7, 22, 25). Assuming that the protonated and deprotonated forms of reduced FdI differ by n protons, the pH dependence of the [3Fe-4S]+/O reduction potential, shown in Fig. 7A, can be described by

Eo',b, = E " a l k + (2.3RT/nF) log(1 + [H+]/&I (1)


where Ed’,,bs is the observed reduction potential, h@la1k is the limiting reduction potential under alkaline conditions, KH is the acid dissociation constant, and other terms have their normal meanings. Nonlinear regression analyses were carried out to fit the Fig. 7A data and the best results are shown in Table I. Best fits (obtained by allowing each of the parameters

-300 A1

-500 - W

V, -750 f vi > > -em E - B -850

-700

0 0 W I .‘

-900

0.. ...... D.. ..?.,,

0‘ ..... -650

“

-750 I I 6 8 9

p7H FIG. 7. Graphs of p’* against pH for redox couples of

AvFdI. All data are for temperature 3 “C, 0.1 M NaCl, mixed buffer, 1-2 mM neomycin or tobramycin. for [3Fe-4S]+” (graph A ) and (4Fe- 4S]2+’1+ (graph C) each datum point is an average for reductive and oxidative resultant peak potentials. For couple C (graph B ) values are for the resultant peak potential measured in the reductive direction at the frequency 30 Hz. Due to interference from couple B in the case of the C20A mutant, only values for native and C24A proteins could be measured. Graph A shows nonlinear regression fit for each set of data to expression (1). Graphs B and C show best linear fits. SHE, standard hydrogen electrode.

Ed’alk , (2.3RT/nF), and KH to float), each gave essentially similar values for pK, Ed’alk and slope -d(h@’,b)/d(pH). The slope value expected for tight coupling to a single H+ is 55 mV at 3 “C. As shown in Table I, the AuFdI values are lower, at -43 mV. At present, we have no explanation for this result. Although we do not yet know the site of protonation, the data in Fig. 7A and in Table I clearly show that the cause for this pH-dependent behavior has not been affected by either mutation.

Further Reduction Associated with the [3Fe-4S] Cluster- In several other ferredoxins that have been examined by voltammetry, the presence of a 3Fe cluster has been associated with a further reduction process at low potential (26). For example, for Desulfovibrio africanus 7Fe FdIII, it has been established that a further two electrons are taken up by the protein, in a complex pH-dependent process, yielding a species with sufficient stability to persist for at least several minutes (27). This reactivity is removed upon transformation of the [3Fe-4S] cluster to a [4Fe-4S] cluster (27). While further two- electron reduction of [3Fe-4SJ0 is the most likely (albeit novel) explanation, the identity of the product has yet to be established. For AuFdI, Fig. 6 shows that like the [3Fe-4S]+’ocouple (peak A), the peak C couple has been unaffected by the C24A mutation. Peak C, unfortunately, overlaps with peak B for the native and C20A proteins. Therefore, no attempts were made at this stage to fit the data for couple C beyond calcu- lating a linear regression line combining data from the native and C24A proteins (Fig. 7B).

[4Fe-4SI2+/‘+ Cluster Potentials-Again, by analogy with the earlier report on AcFdI, peaks B of Fig. 6 are assigned to the one-electron reduction and reoxidation of the [4Fe-4SI2+ cluster. The value obtained for the nativeAvFd1 protein (-648 mV at pH 8.1) is in close agreement with the earlier value (-645 mV at pH 8.3) reported for AcFdI (18) and also confirms our prior conclusion that the AvFdI potential is more negative than -500 mV (20). Peak positions B differ widely among the three forms of the protein. The results are quantitated in Fig. 7C which is a plot of the [4Fe-4SI2+/’+ potential for all three proteins as a function of pH. Reduction potentials for the C24A mutant are -50 mV more positive than for the native protein while potentials for the C20A mutant are -100 mV more negative. The data in Fig. 7C also show a small pH dependence of Eo’ for this couple, and values for d(Ed’,~,)/

TABLE I Reduction potentials for the [Fe-SI clusters of native, CZOA, and C24A forms of AvFdI

Couple Protein Eo’,b. (pH 7.8)” -d(E)/d(pHh.b -d(E)/d(pH)limc PK’ E O ’ d k

mV mV mV mV [3Fe-4S]’+/0 (=A) Native -425 (f5) 43 (+4) 7.8 (+0.2) -445 (+15)

C20A C24A

-429 ( f 5 ) 42 (+3) 7.6 (+0.2) -440 (+15) -427 (k5) 43 (+4) 7.9 (+0.2) -449 (+15)

[4Fe-4S]’+/’+ (=B) Native -647 (f5) 16 (0.96) C20A -746 ( f l0 ) 15 (0.95) C24A -600 ( f5) 18 (0.92)

C d Native C24A

-772 (f15) -782 (k5) 52 (0.98)

Eo’ values for couples A and B were determined from the average of scans made in the reductive and oxidative directions over the frequency range 15-80 Hz. Eo’ values for couple C were obtained from the reductive direction only since signals tended to be better defined. Temperature 3 “C for all measurements. All values are given versus standard hydrogen electrode. Estimated errors are shown in parentheses.

* Estimated from linear regression analysis. ‘Best fit (nonlinear regression) to expression (1) in text, no parameters fixed. Values in parentheses are

Couple C of Fig. 6 may be the couple [3Fe-4SI0/’- although other interpretations are possible. The identity of correlation coefficients.

this “hyper-reduced” species, formally at the all-Fe(I1) level, is currently under investigation.


d(pH) are included in Table 1. Although we do not, at present, know the cause for this pH behavior, all three slopes in Fig. 7C are the same, and thus, we conclude that whatever the cause, it has not been affected by either mutation.

The data shown in Figs. 6 and 7 demonstrate that the reduction potential of the [4Fe-4S]2’’1+ couple varies over a 150 mV range, without change in the potential of the [3Fe- 4S]”/O couple. The two clusters are 11 A apart center-to- center and 6.7 apart at their closest points of contact in native FdI ( 5 ) . The greater variation of the [4Fe-4S] cluster potential is not surprising since the mutation-induced structural changes are much greater in the neighborhood of this cluster. The potential in C24A is -50 mV higher than in native FdI (the corresponding alteration in free energy change is -1.1 kcal mol-‘). Since the structures of native FdI and C24A are extremely similar, with the exception of the replacement of Cys-24 with alanine, the difference in potential may reflect a direct interaction of Cys-24 with the [4Fe-4S] cluster in native FdI. In particular, if Cys-24 is ionized throughout the pH range of this study, its negative charge would lower the reduction potential of the [4Fe-4S] (CYS)~ cluster whose net charge changes from -2 to -3. Alternatively, the difference may be due to the increased space available to the cluster in C24A, favoring the larger, reduced [4Fe-4S]+ cluster (28).

The reduction potential of the [4Fe-4S] cluster in C20A is -100 mV lower than in native FdI ((A,AG) -2 kcal mol-’). Since this change is opposite in direction to that in C24A, the consequences of removing a neighboring cysteine are appar- ently outweighed by the other structural differences between native FdI and C20A. At this time we cannot analyze this change in greater detail. It is possible that the difference in torsional angles of Cys-20 in native FdI and of Cys-24 in C20A is relevant. It is also noteworthy that a significant change in hydrogen bonding occurs from native FdI to C20A, although the number, type, and distance of the NH. . -S hydrogen bonds to the [4Fe-4S] cluster itself are the same. An examination of the surface accessibility of the [4Fe-4S] clusters of native FdI, C20A, and C24A has not detected any significant differences (IO), and it therefore seems unlikely that the reduction potentials are changed by differential sol- vent contributions.

Fe(CN)i- Oxidation-In addition to reversible redox behavior, [Fe-S] clusters may be irreversibly oxidized by strong oxidants. The lability of many [Fe-SI proteins in the presence of O2 is attributable to such degradative reactions. Native FdI is unaffected by O2 but does react with Fe(CN)i- in a three- step degradative process (20,29,30). The first step is a three- electron oxidation involving the [4Fe-4S] cluster, which gives rise to a paramagnetic species. This exhibits EPR up to quite high temperatures and is most easily monitored in the range 40-60 K, where the [3Fe-4S]+ cluster EPR is not detectable. In contrast to the latter cluster, the paramagnetic product of Fe(CN)i- oxidation exhibits no measurable visible-near UV magnetic circular dichroism (20). This observation led to the proposal that it is formed by dissociative oxidation of the [4Fe-4SI2+ cluster, involving a one-electron oxidation of one cysteine ligand and a two-electron oxidation of one S2- to give a cysteinyldisulfide (Cys-S-S) radical. The more recent structural data now suggest that the participating cysteine might not be a cluster ligand, but rather the adjacent cysteine (Cys- 241, whose Sy is extreme1y:lose to the [4Fe-4S] cluster (the distance to one S2- is 3.35 A) ( 5 ) . The C24A and C20A variants of FdI, which both lack an adjacent cysteine, permit further elucidation of this reaction.

Fig. 8 shows the EPR spectra resulting from addition of -3.0 equivalents of Fe(CN)i- to native, C24A, and C20A FdIs.

I

H- -9

FIG. 8. EPR spectra of native FdI (A and B ) , C24A (C and D), and C20A ( E and F) treated with 3.0 equivalents of Fe(CN)$- (incubation times 65 min. The microwave frequency was 9.6 GHz; the microwave amplitude was 5 G; the microwave powers were 0.39 mW (A), 1 mW ( B , C, and E ) , and 2 mW (D and F ) ; the protein concentrations were 40 pM ( A and B ) , 74 pM (C and D), and 61 @M ( E and F ) ; and the gains were 2.5 X lo4 (A), 4 X 10’ ( E ) , 1.6 X lo4 (C), 1 X lo5 (D andF), and 1.24 X lo4 ( E ) . A, C, and E were recorded at -10 K, while B, D, and F were recorded in the range 40-50 K.

At -10 K, the EPR of native FdI, attributable to the [3Fe- 4S]+ cluster, is broadened and becomes more intense on addition of Fe(CN)i- (Fig. 8A). At -40 K, the EPR of the paramagnetic Fe(CN):- oxidation product is observed (Fig. 8B). In both C24A and C20A, the -10 K spectra are unaffected in shape and intensity by Fe(CN)i- (Fig. 8, C and E ) and no EPR is detectable at - 40 K (Fig. 8, D and F).

These results suggest strongly that the formation of the paramagnetic Fe(CN)i- oxidation product in native FdI re- quires the presence of an adjacent cysteine. This in turn is consistent with that species being 24Cys-S-S’. The formation of this radical species has not been characterized in any other [Fe-SI protein. Fig. 1 shows that six other 7Fe ferredoxins have an analogous Cys at position 24. Studies of Fe(CN)i- oxidation, however, have been reported only for the Pseudo- m o m ovalis, Mycobacterium smegmatis, and Thermus thermophilus ferredoxins. For these proteins, NMR studies (31, 32) have led to the conclusion that Fe(CN)i- oxidizes the [4Fe-4S] cluster to a [3Fe-4S] cluster. However, in an inde- pendent study of T. thermophilus ferredoxin no reaction was detected (25). It therefore appears that the sequence proximity of Cys-24 to Cys-20 is necessary, but not sufficient, to guar- antee the formation of the paramagnetic Fe(CN)i- oxidation product.

For the native protein, further addition of Fe(CNI3- 6 causes complete destruction of the [4Fe-4S] cluster followed by destruction of the [3Fe-4S] cluster (20,29,30). For comparison, the effects of addition of up to 30 equivalents Fe(CN)i- on the visible-UV absorption, CD and EPR of C24A and C20A


are summarized in Figs. 9 and 10. In the case of C24A, no reaction is observed (within experimental error). Thus, the formation of the disulfide radical appears to be a prerequisite for the subsequent destructive reactions. In the case of C20A, at low Fe(CN)2- to protein ratios no reaction is apparent, but the absorption and CD spectra show evidence of some reaction as the ratio increases (Fig. 10B). The percentage change in the CD is wavelength dependent (Figs. 9B and 10B), suggest-

' x)3 350 403 450 333 550 Mx) 650 I I I I I I I I I

rlnml

FIG. 9. Circular dichroism spectra of C24A (A) and C20A ( B ) treated with 20 equivalents of Fe(CN)z- for 50 min (lower trace in each panel) compared with untreated protein (upper trace in each panel). The protein concentrations were 74 p~ in A and 60 p~ in B.

120, * I

0

B

0 10 20 30 Equivalents Fe(CN)c3-

FIG. 10. Fe(CN):- titration of C24A ( A ) and C20A ( B ) . The results are expressed as a percentage of absorption, circular dichroism, and EPR signal intensities remaining after treatment with 0-30 equivalents of Fe(CN)t-. Absorption, CD, and EPR data are for -45, -50, and -60 min incubation times, respectively. The absorption points are a t X = 500 nm, the absorption from peak unreacted Fe(CN)i- is negligible. The CD points are to trough intensities for the major features of the spectra as indicated. The EPR points are integrated intensities of the g -2.01 signal a t 10 K, 1 mW microwave power, 9.59 GHz microwave frequency, and 5 G modulation amplitude. Background EPR from unreacted Fe(CN)$- was subtractedprior to integration.

. * ,

a b c

FIG. 11. In vivo the C24A protein accumulates to the same levels as native AvFdI, while the C20A protein accumulates to much lower levels. As shown in Fig. 2, the three strains used, JGlOO/pDB214, JGIOO/pA VlOO, and JGlOO/pJG4 overproduce native FdI ( A ) , C24A ( B ) , and C20A (C), respectively. Shown is sodium dodecyl sulfate gel electrophoresis separation of cell-free extracts from each strain after blotting and reacting with anti-FdI antibodies. In all cases 120 pg of protein was loaded/lane.

ing that the reaction is affecting the [Fe-SI clusters unequally. Because the [3Fe-4S]+ EPR is unaffected by Fe(CN)2- (Fig. 10B) it appears that the reaction is occurring preferentially at the [4Fe-4S] cluster. Since the absorption is diminished and no new EPR appears near g - 2, the product of this reaction cannot be either a [4Fe-4SI3+ cluster or a [3Fe-4S]' cluster. Most probably, therefore, the reaction leads to complete destruction of the [4Fe-4S] cluster.

Stability in Vivo-The structural rearrangement caused by the C20A mutation also leads to a protein with greatly decreased stability in uiuo, whereas the C24A protein appears to be as stable as native FdI. This is illustrated in Fig. 11 which shows the one-dimensional sodium dodecyl sulfate-gel electrophoresis separation of proteins in cell-free extracts of A. uinelandii strains, after blotting and reacting with anti-FdI antibodies. The C24A protein appears to accumulate to the same levels as the native while the C20A protein accumulates to much lower levels, which we estimate to be -10% of native. The relative levels of the three proteins is also observed in vivo by blotting colonies followed by reaction with anti-FdI antibodies (data not shown). These results are not simply due to differing reactions to the antibodies because the final yields from protein purification give the same trends. Thus, a preparation from 1000 g of A. uinelandii cells (wet weight) yields -400 mg of purified native FdI or purified C24A while the same sized preparation of the C20A overproducing strain gives only -40 mg of protein.

We currently believe that the lowered level of the C20A protein accumulated in uiuo is due to lowered protein stability, rather than decreased initial synthesis, because the same phenomenon is observed with the three different expression systems described under "Experimental Procedures." Thus, the levels of the C20A protein accumulated are always -10% of native when the protein is expressed from its normal location on the chromosome, from behind the nifH promoter or from its own promoter in pKT230. At present, we cannot determine if this proteolysis problem occurs during protein folding or after the protein is assembled into its final structure. However, we suspect the former because the folded protein appears to be as stable as the native during cell rupture, purification, and crystallization. As indicated above, once purified, it is also as stable as the native toward 02 and only slightly less stable toward destruction by Fe(CN)2- than the C24A protein.

Ability to Function in A. uinelandii Metabolism-The spe- cific function of FdI in A. uinelandii is currently unknown. It was long believed to serve as the immediate electron donor to nitrogenase, at least under some physiological conditions (33). Such a view was recently dispelled, however, when the fdxA gene, which encodes AuFdI, was cloned and sequenced (15).


TABLE I1 Growth rates of A. vinelandii strains expressing different

variants of FdI

Strain FdI Fld Doubling time” Ref.

h Wild type Native + 2.3 16 LMlOO + 2.3 16 DJ130 Native - 2.3 16 DJ138 - 5.3 16 JG103 C20A - 2.3 This study JG118 C24A - 2.3 This study

’ Doubling times were measured in liquid culture on Burk’s plus ammonium acetate as described under “Experimental Procedures.” All strains exhibited biphasic growth on this medium utilizing acetate as the first carbon source and then utilizing sucrose. Only growth on sucrose is shown but trend is the same for growth on acetate. Key: Fld, flavodoxin. Genotypes of strains JG103 and JG118 are shown in Fig. 2.

FIG. 12. Native, C20A. and C24A FdIs, restore wild type colony morphology to FdI-/Fld- strain, DJ138. Plates containing Burk’s medium plus ammonia were inoculated with various strains of A. vinelandii and photographed after 6 days a t 30 “C. All four strains are missing flavodoxin (Fld) but differ in the type of FdI they contain. Panel A shows Fld- strain DJ130 which synthesizes native FdI and is representative of the larger uniform colony morphology also exhibited by wild type and FdI- strains (16). Panel B (DJ138) shows the small variable colony morphology of the FdI-Fld- strain (16). Panels C (JG103) and D (JG118) show the restoration of the large uniform morphology in CZOA/Fld- and C24A/Fld- strains.

That study showed that the fdxA gene was not located in the N2 fixation ( n i f ) region of the A. uinelandii chromosome, that FdI expression was not coregulated with nitrogenase, and that elimination of FdI synthesis in uiuo, by disruption of the fdxA gene, did not alter the N2 fixing ability of the cells. A parallel study of another putative electron donor to nitrogenase, flavodoxin (Fld), showed that it was the immediate electron donor in A. uinelandii (34). Thus, Fld is encoded by the nifF gene which is located in the N2 fixation region of the chromosome, and its expression is coregulated with nitrogenase (although a small amount of this protein is also constitutively expressed). Therefore, it was surprising to find that elimination of Fld synthesis in viuo, by disruption of nifF, also did not alter the N2 fixing ability of the cells (34). These results led us to construct a double FdI-/Fld- strain which we dem- onstrated was still able to fix N2 at wild type rates (16).

Further characterization of the double FdI-/Fld- strain, DJ138, led us to an additional observation, unrelated to N2 fixation, which we have exploited in this study to test whether or not the C20A and C24A variants of FdI are functional in A uinelandii cells. As shown in Table 11, in liquid media, the FdI- strain JGlOO and the Fld- strain DJ130 both grow at wild type rates in the presence of NH: whereas, the FdI-/ Fld- double mutant strain DJ138 grows much more slowly (16). These data demonstrate that there is some metabolic

function, unrelated to N P fixation, that is normally carried out by FdI or Fld and that if one of these proteins is missing, the other can substitute for it. Because A. uinelandii has a high frequency reciprocal recombination system, we are now able to replace the missing FdI protein in the double FdI-/ Fld- strain DJ138 with the C20A or C24A versions of FdI. If the mutant proteins are functional in vivo then the resultant strains should grow a t wild type rates, while if they are not functional, the resultant strains should continue to grow at the slower DJ138 rate.

The data in Table I1 show that the double C20AFdIFld- and C24AFdIFld- strains do grow at wild type rates. Thus, in this in uiuo assay both mutant proteins are able to function as well as the native protein. I t is important to note that the reduction potential for the [4Fe-4S] cluster in native FdI (approximately -650 mV) was the lowest known for a non- photosynthetic biological system. The observation that the C20A FdI protein (whose [4Fe-4S] reduction potential is -750 mV) is functional in uiuo, leads to the questions: is the [4Fe- 4SI2+/’+ cluster physiologically relevant and, if so, how is it reduced in uiuo? These issues are under further investigation.

A second observation that we have made concerning the growth of the FdI-/Fld- strain DJ138, is illustrated in Fig. 12. These data show that the wild type, FdI- and Fld- strains all produce large colonies on solid media and that the colonies are of uniform size. In contrast, the slower growing FdI-Fld- strain never produces colonies as large as the wild type. In fact, as shown in Fig. 12, the DJ138 colonies are both small and variable in size. Measurements of the colony sizes indicate that there is a continuous gradation of sizes from the smallest to the largest, rather than two distinct populations. This is also confirmed by the observation that repeated streaking of either the smallest or the largest DJ138 colonies, always yields the original variable-sized population. Fig. 12 shows that both the C20A and C24A versions of FdI are able to restore the large uniform colony morphology exhibited by the wild type strain when they are expressed in a Fld- background. Thus, in spite of their altered [4Fe-4S] cluster reduction potentials, both proteins appear to be able to function in A. uinelundii metabolism.

Ability to Repress the Synthesis of Protein X-In previous studies we have shown that FdI “represses” the synthesis of an -18,000 M, protein which we have designated protein X (16). At present, we do not know if this “repression” is direct or indirect and mechanistic studies are in progress. This FdI function is illustrated in Fig. 13 which shows two-dimensional gel electrophoresis separations of proteins in cell-free extracts of a number of A. uinelundii strains. As illustrated in panel A of Fig. 13, the wild type strain produces only tiny amounts of protein X while panel B shows that the protein is dramatically overproduced in strain LMlOO which does not synthesize FdI, due to disruption of the fdxA gene (16). Panel C shows that a strain which is identical to wild type except that it synthesizes C24AFdI (instead of native FdI) contains only tiny levels of protein X. Thus, like native FdI, C24AFdI can repress the synthesis of protein X. In contrast, panel D shows that when C20A FdI is synthesized from its normal location on the chromosome, protein X synthesis is not repressed. Because the C20A protein is present in only 10% wild type levels in this strain (see above) we suspect that its inability to repress protein X synthesis might be related to the amount of C20A protein present rather than to its structure or reduction potential. This view is confirmed in Fig. 13, panel E, which shows that overproduction of the C20A protein in pKT230 does lead to repression of protein X synthesis. Thus again, both the C24A and C20A proteins appear to function like

Site-directed Mutagenesis: AvFdI 21571 " ." 10. Soman, J., Iismaa, S. & Stout, C. D. (1991) J. Biol. Chem. 266 ,

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14. Taylor, J. W., Ott, J. & Eckstein, F. (1985) Nucleic Acids Res.

15. Morgan, T. V., Lundell, D. J. & Burgess, B. K. (1988) J. Biol. Chem. 263,1370-1375

16. Martin, A. E., Burgess, B. K., Iismaa, S. E., Smartt, C. T., Jacobson, M. R. & Dean, D. R. (1989) J. Bacteriol. 171,3162- 3167

17. Ditta, G., Stanfield, S., Corbin, D. & Helinski, D. H. (1980) Proc. Natl. Acud. Sci. U. S. A. 7 7 , 7347-7351

18. Armstrong, F. A., George, S. J., Thomson, A. J. & Yates, M. G.

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FIG. 13. Like native FdI, C20A, and C24A can also repress 20. Morgan, T. V., Stephens, P. J., Devlin, F., Stout, C. D., Melis, K. the synthesis of protein X. Protein X is identified on Coomassie- A. & Burgess, B. K. (1984) Proc. Natl. Acud. Sci. U. S. A. 8 1 , stained two-dimensional gel electrophoresis separations of cell free extracts as described previously (15). A. vinelandii strains used are: 21. yoch, D. c. & Carithers, R. p. (1978) J. Bacterial. 136,822-824 wild-type ( A ) , FdI- strain LMlOO ( B ) (15), C24A strain JG106 (c), 22. George, S. J., Richards, A. J. M., Thornson, A. J. & Yates, M. G. C20A strain JGZOA (D), and C20A overproduction strain JG100/ pJG4 ( E ) .

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547-556

1931-1935

native FdI in this in vivo assay. Conclusions-We have applied site-directed mutagenesis to

the study of a crystallographically characterized [Fe-SI protein. Here we have begun to correlate changes in structure with changes in [Fe-SI redox properties, chemical reactivity, and function by examining the C20A and C24A proteins. By continued examination of additional structurally characterized mutants, using spectroscopic, electrochemical, and bio- chemical methods we should ultimately be able to predict changes in [Fe-SI cluster redox properties and chemical reactivity based on sequence and other structural information.

Acknowledgments-We wish to thank Dr. C. David Stout for help- ful discussions, Dr. Arthur Sucheta for computer analysis of electrochemical data, and the Exxon Education Foundation for an award (to F. A. A.).

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site-directed mutagenesis of azotobacter vinelandii ferredoxin i

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