©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modified Ligands to F and F in Photosystem I
I. STRUCTURAL CONSTRAINTS FOR THE FORMATION OF IRON-SULFUR CLUSTERS IN FREE AND REBOUND PsaC (*)

(Received for publication, April 6, 1995; and in revised form, August 3, 1995)

Tetemke Mehari (1)(§) Fengyu Qiao (2) M. Paul Scott (2)(¶) David F. Nellis(¶) (3) Jindong Zhao (4) Donald A. Bryant (4) John H. Golbeck (2)(**)

From the  (1)Department of Chemistry, Addis Ababa University, Addis Ababa, Ethiopia, the (2)Department of Biochemistry and Center for Biological Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, the (3)Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, and the (4)Department of Biochemistry and Molecular Biology and Center for Biomolecular Structure and Function, Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cysteines 14, 21, 34, 51, or 58 in PsaC of photosystem I (PS I) were replaced with aspartic acid (C21D and C58D), serine (C14S, C34S, and C51S), and alanine (C14A, C34A, and C51A). When free in solution, the C34S and C34A holoproteins contained two S= ground state [4Fe-4S] clusters; all other mutant proteins contained [3Fe-4S] clusters and [4Fe-4S] clusters; in addition, there was evidence in C14S, C51S, C14A, and C51A for high spin (S = ) [4Fe-4S] clusters, presumably in the modified site. These findings are consistent with the assignment of C14, C21, C51, and C58, but not C34, as ligands to F(A) and F(B). The [4Fe-4S] clusters in the unmodified sites in C14S, C51S, C14A, and C51A remained highly electronegative, with E(m) values ranging from -495 to -575 mV. The [3Fe-4S] clusters in the modified sites were driven 400 to 450 mV more oxidizing than the native [4Fe-4S] clusters, with E(m) values ranging from -98 mV to -171 mV. A C14D/C51D double mutant contains [3Fe-4S] and S= [4Fe-4S] clusters, showing that the 3Cysbullet1Asp motif is also able to accommodate a low spin cubane. When C34S, C34A, C14S, C51S, C14A, and C51A were rebound to P700-F(X) cores, electron transfer to F(A)/F(B) was regained, but functional reconstitution has not yet been achieved for C21D, C58D, or C14D/C51D. These data imply that PsaC requires two iron-sulfur clusters to refold, one of which must be a cubane. Since two [4Fe-4S] clusters are found in all reconstituted PS I complexes, the presence of two cubanes in free PsaC may be a necessary precondition for binding to P700-F(X) cores.


INTRODUCTION

PsaC in photosystem I (PS I) (^1)shares similarities with a number of soluble 2[4Fe-4S] ferredoxins in terms of molecular mass, amino acid sequence, and the presence of two CXXCXXCXXXCP iron-sulfur cluster binding motifs. The protein has greatest similarity to the amino acid sequence of Chromatium vinosum ferredoxin, for which no three-dimensional structure is available. However, there is enough sequence homology with the 54-amino acid 2[4Fe-4S] ferredoxin from Peptococcus aerogenes(1) and the first 58 residues of the 106-amino acid [3Fe-4S]bullet[4Fe-4S] ferredoxin from Azotobacter vinelandii(2) to make predictions of tertiary structure based on the known three-dimensional structure of these proteins (Fig. 1). Assuming that the overall folding of the PsaC polypeptide backbone is analogous to these known structures, cysteines 11, 14, 17, and 58 should provide the ligands to one [4Fe-4S] cluster, and cysteines 21, 48, 51, and 54 should provide the ligands to the second [4Fe-4S] cluster. The two clusters in PsaC should also be related by a pseudo-2-fold axis of symmetry, so that cysteines 11, 14, 17, and 58 correlate with cysteines 48, 51, 54, and 21, respectively.


Figure 1: Backbone structure of PsaC based on modeling studies with P. aerogenes (adapted from (19) and (20) ) with the proposed assignment of cysteines based on experimental findings in (5) . The similarity between the two proteins is highlighted by the x-ray crystal structure of the cyanobacterial PS I reaction center, which shows that the distance between the iron-sulfur clusters is 12 Å, identical to the intercluster distance in P. aerogene ferredoxin. An extra 10 amino acids are required between the two iron-sulfur binding motifs, and an extra 14 amino acids are required on the C terminus for accurate alignment. The cysteines chosen for site-directed mutagenesis (C14D, C14S, C14A, C21D, C51D, C51S, C51A, and C58D) are identified; in addition, a double and triple mutant (C14D/C51D and C14D/C51D/C34S) were generated. Cysteine 34 lies in the loop region between cysteines 21 and 48. Iron-sulfur clusters F(A) and F(B) were identified (5) by their g values and response to illumination in C14D- and C51D-rebound PS I complexes.



The sequences of naturally occurring ferredoxins suggest modifications that may be tolerated in PsaC. For example, several naturally occurring ferredoxins contain aspartic acid at the position analogous to cysteine 14 in PsaC. These proteins are capable of supporting a mixed spin (S = and ) [4Fe-4Sbullet3Cys1Asp] cluster in the modified site(3, 4) . Using site-directed mutagenesis, mutant PsaC proteins have been constructed with similar mutations (PsaC-C14D and PsaC-C51D), and the free proteins were found to contain [3Fe-4S] clusters at the modified sites and [4Fe-4S] clusters at the unmodified sites(5, 6) . When rebound to PS I cores, the g values and reduction behavior of the [4Fe-4S] clusters in the unmodified sites indicated that F(B) is ligated by residues 11, 14, 17, and 58 and that F(A) is ligated by residues 21, 48, 51, and 54(5) . A ninth cysteine is located at position 34 in all PsaC proteins sequenced thus far, and based on the structural analogy with P. aerogenes, this residue is not predicted to be involved in ligation of either cluster.

The goal of this study is to determine the effect of cysteine replacements on the refolding of PsaC and on the rebinding of modified PsaC proteins to P700-F(X) cores. Mutant PsaC proteins were constructed in which one of the cysteine ligands to an iron-sulfur cluster was changed to the charged amino acid aspartate (PsaC-C14D, previously reported in (6) , and PsaC-C51D, in the accompanying paper(36) ), the polar amino acid serine (PsaC-C14S and PsaC-C51S), or the neutral amino acid alanine (PsaC-C14A and PsaC-C51A). Modifications not previously known in ferredoxins (PsaC-C21D and PsaC-C58D) were also made to determine whether a [3Fe-4S] cluster could be accommodated when the proline-proximal cysteine in either cluster-binding motif is altered. In an effort to determine whether cysteine 34 is a ligand to a cubane iron, serine (PsaC-C34S) and alanine (PsaC-C34A) mutations were constructed and characterized at this site. Finally, novel double and triple mutants containing one aspartate in both iron-sulfur cluster binding sites (PsaC-C14D/C51D and (PsaC-C14D/C51D/C34S) were constructed. The mutant proteins were studied for their ability to ligate a [3Fe-4S] or a [4Fe-4S] cluster, to rebind to P700-F(X) cores, and to reestablish electron transfer to F(A) and F(B).


MATERIALS AND METHODS

Engineering, Synthesis, and Purification of the Mutant PsaC Protein

All PsaC mutant proteins were produced from derivatives of the pET expression plasmid pET-36C, which contains the psaC gene isolated from Synechococcus sp. strain PCC 7002(7) . Site-directed mutagenesis was performed as described previously(5, 8) . Escherichia coli BL2(DE3) harboring pET-36C and derivatives (psaC and mutant psaC) were grown in NYZCM medium (9) except that magnesium sulfate was omitted. The growth medium for cells synthesizing PsaC proteins was supplemented with 6 mg ml ferric ammonium citrate. Expression was performed as described previously (10) and was initiated by the addition of isopropyl-1-thio-beta-D-galactopyranoside to a final concentration of 0.5 mM to the growth medium. After 1 h, 20 µg ml rifamycin was added to the medium, and expression was continued for an additional period of 5-7 h. Cells were harvested by centrifugation and washed once with TS buffer (20 mM Tris-HCl, pH 8.0, and 10 mM NaCl). The cells were resuspended in TS buffer containing 2 mM dithiothreitol and 1 µg ml DNase and disrupted by two passes through a French pressure cell at 18,000 p.s.i. at 4 °C. Inclusion bodies were collected by centrifugation of the whole-cell extract at 7650 times g for 10 min at 4 °C. The inclusion bodies were solubilized with 7 M urea and 2 mM dithiothreitol in 50 mM Tris, pH 8.3, and 2 mM EGTA. The solubilized proteins were purified by gel exclusion chromatography over Sephadex G-75 and eluted with 2 mM dithiothreitol and 50 mM Tris-HCl, pH 8.3. Two protein bands were separated; the light brown band (6) was collected, analyzed by SDS-polyacrylamide gel electrophoresis and subjected to N-terminal amino acid sequencing to verify the presence of the amino acid substitution at the expected position.

Purified PsaC was refolded with iron-sulfur clusters as described (7) and purified by ultrafiltration in an Amicon cell over a YM-5 membrane (Amicon, Beverly, MA) with 50 mM Tris-HCl, pH 8.3, and 0.1% beta-mercaptoethanol. For electrochemistry, the PsaC holoproteins were desalted, and the beta-mercaptoethanol was removed by gel filtration chromatography over a Sephadex G-25 (Pharmacia Biotech Inc.) column under anaerobic conditions in a Coy controlled environment chamber (Grass Lake, MI).

Purification of Expressed PsaD and PsaE

Nostoc sp. strain PCC 8009 PsaD (7) was purified from solubilized inclusion bodies by chromatography on CM-Sepharose CL-6B (Sigma) with a linear gradient of sodium chloride (50-1000 mM). Synechococcus sp. strain PCC 7002 PsaE (11) was purified from solubilized inclusion bodies by chromatography on DEAE-Sepharose CL-6B (Sigma) with a linear gradient of sodium chloride (10-200 mM) at pH 8.0.

Protein Analysis

Protein concentrations were determined using a dye-binding method(12) . The Bradford dye binding assay, when based on bovine globulin as standard, overestimates the amount of PsaC apoprotein by a factor of 1.43 and the PsaD protein by factor of 1.85(7) . N-terminal amino acid sequencing of the overproduced mutant proteins was performed with a Milligen/Biosearch 6600 ProSequencer at the University of Nebraska Protein Core Facility. The protein samples were dialyzed against water, and aliquots were subjected to automated Edman degradation after being immobilized on arylamine membrane disks (Sequelon-AA, Milligen/Biosearch, Burlington, MA). The mutant proteins were diluted with 50% ethanol, 2% acetic acid (v/v) and dried on arylamine disks at 55 °C. Covalent attachment to the membrane was performed using water-soluble carbodiimide in MES buffer at pH 5.0. The arylamine disks containing covalently attached protein were washed with methanol, water, and methanol prior to sequencing. Proteins with substitutions in positions 14 and 21 were determined by direct sequencing from the NH(2) terminus. Proteins with substitutions in positions 34, 51, and 58 were determined from an internal tryptic peptide purified by reverse-phase HPLC.

Reconstitution of Mutant PsaC onto P700-F(X)Cores

The method for resolution and reconstitution of the PS I complex is described(13) . Briefly, the P700-F(X) cores are prepared from PS I complexes isolated from Synechococcus sp. PCC 6301 by treatment with 6.8 M urea until the 30-ms back reaction between P700 and [F(A)/F(B)] is replaced by a 1-ms back reaction between P700 and F(X) (a process typically requiring between 20 and 25 min). The reaction mixture is quenched by 100-fold dilution with 50 mM Tris, pH 8.3, dialyzed further in the same buffer, and concentrated by ultrafiltration over a YM-100 membrane (Amicon, Beverly, MA). Reconstitutions are performed by adding 10 mg of PsaC apoprotein, 20 mg of PsaD, and 10 mg of PsaE to 2 mg (total chlorophyll) of the P700-F(X) core. The ratio of PsaC:PsaD:PsaE:PS I core is approximately 15:15:15:1. The iron-sulfur clusters are rebuilt by the addition of 0.5 ml beta-mercaptoethanol, 0.5 ml of 30 mM FeCl(3), and 0.5 ml of 30 mM Na(2)S to 100 ml of reconstitution medium, waiting 20 min between each addition, and incubation overnight at 4 C. The free proteins are removed, and the reconstituted PS I complex is concentrated by ultrafiltration over a YM-100 membrane.

EPR Spectroscopy

X-band EPR spectroscopy was performed using a Bruker ECS 106 spectrometer operating with a normal mode (ST 8615) or a dual mode (DM 4116) resonator. Temperatures were controlled using an Oxford Intelligent temperature controller with liquid nitrogen as the reference. PsaC was approximately 2.5 mg ml. Chemical reduction was carried out by adjusting the pH to 10.5 with 1 M glycine-NaOH and adding approximately 1 mg of sodium dithionite/300 µl of sample. All manipulations of the PsaC holoprotein were carried out under anaerobic conditions. The midpoint potentials of the mutant PsaC proteins were determined using electrochemical poising followed by EPR detection ( (6) and references therein). Redox dyes used for the titration were as follows: N-N`-trimethylene-2,2`-dipyridinium dibromide (E(m) of -540 mV), 4,4`-dimethyl-N-N`-trimethylene-2,2`-dipyridinium dibromide (E(m) of -680 mV), methyl viologen (E(m) of -440 mV), thionine (E(m) of +56 mV), indigotetrasulfonic acid (E(m) of -46 mV), indigodisulfonic acid (E(m) of -125 mV). The first two dyes were synthesized as described(14) . All other dyes were purchased from Aldrich.


RESULTS

Substitutions in the Second Cysteine in the CXXCXXCXXXCP Motif of PsaC: EPR Spectra of [3Fe-4S] and [4Fe-4S] Clusters in C14S, C51S, C14A, and C51A

The EPR spectra of free PsaC-C14S, PsaC-C51S, PsaC-C14A, and PsaC-C51A were found to be similar to those observed previously for the PsaC-C14D and PsaC-C51D mutant proteins(6) . With no additions, the EPR spectra show a peak at g = 2.020 and trough at g = 1.998, characteristic of oxidized [3Fe-4S] clusters (Fig. 2, dotted lines), and the resonances disappear after mild chemical reduction with sodium dithionite at pH 8.0 (E(m) = -480 mV). When the samples are more strongly reduced by the addition of sodium dithionite at pH 10.5 (E(m) = -630 mV), the EPR spectra show a near axial set of resonances with broad line shapes and a g of 1.94, characteristic of reduced [4Fe-4S] clusters (Fig. 2, solid lines). The line shapes of the merged midfield and highfield resonances of PsaC-C14S and PsaC-C14A (Fig. 2, A and C) are slightly broader than the corresponding features in PsaC-C51S and PsaC-C51A (Fig. 2, B and D), a pattern observed earlier for PsaC-C14D and PsaC-C51D(6) . These features are consistent with the presence of an S= ground state [4Fe-4S] cluster, identified as F(A) in PS I-rebound PsaC-C14D, and by the presence of a S= ground state [4Fe-4S] cluster, identified as F(B) in PS I-rebound PsaC-C51D.


Figure 2: EPR spectra of the oxidized [3Fe-4S] (dotted line) and reduced [4Fe-4S] (solid line) clusters in free PsaC-C14S (A), PsaC-C51S (B), PsaC-C14A (C) and PsaC-C51A (D) mutant proteins. The proteins were oxidized by brief exposure to air at pH 8.3 and reduced in the presence of sodium dithionite at pH 10.5. Signal intensity was measured in a matrix of temperature and microwave power to determine the temperature optimum and half-saturation parameter, P, for each cluster. Spin quantitation was performed at the temperature optimum and at power settings an order of magnitude below the P value. The vertical axis shows signal intensity with the reduced [4Fe-4S] cluster scaled arbitrarily to unity spin concentration; scaling factors of 0.25 (A), 2.0 (B), 1.0 (C), and 0.25 (D) were applied to the [3Fe-4S] clusters. Spectrometer conditions were as follows: standard mode resonator, microwave power, 10 mW for the [4Fe-4S] clusters, 1 mW for the [3Fe-4S] clusters; microwave frequency, 9.456 GHz; modulation amplitude, 10 G at 100 kHz; temperature, 15 K for the [4Fe-4S] clusters and 35 K for the [3Fe-4S] clusters.



The temperature optimum of the [3Fe-4S] clusters in PsaC-C14S, PsaC-C14A, PsaC-C51S, and PsaC-C51A is 30 K (determined at 5 mW of microwave power). The temperature optima of the [4Fe-4S] clusters in PsaC-C14S and PsaC-C51S are 12 and 9 K, and in PsaC-C14A and PsaC-C51A they are 12 and 15 K (determined at 20 mW of microwave power). The overall pattern is that the presence of a charged, polar, or hydrophobic amino acid result in small differences in the spin relaxation properties, as inferred from the temperature optimum and half-saturation parameter P, of the [3Fe-4S] clusters in the modified sites or in large changes in the relaxation properties of the [4Fe-4S] clusters in the unmodified sites (data not shown).

To demonstrate that the [3Fe-4S]^0 clusters are indeed present in these proteins (i.e. that chemical reduction does not result in their destruction), the four mutant proteins were analyzed by perpendicular and parallel mode EPR. A reduced [3Fe-4S] is detectable because it is paramagnetic with a ground state spin S= 2, and can be observed in normal mode EPR as a single, asymmetric resonance at g = 10-12, which extends into zero field. Under mildly oxidizing conditions, we were unable to detect any significant resonances in PsaC-C14A, PsaC-C51A, PsaC-C14S, or PsaC-C51S between 0 and 100 mT (data not shown). When the samples were treated with sodium dithionite at pH 10.5 to reduce both the [3Fe-4S] and [4Fe-4S] clusters, the reduced [3Fe-4S]^0 clusters were easily observed around g = 10-12 at high microwave powers and at very low temperatures (Fig. 3, solid lines). The reduced PsaC-C14D (34) and PsaC-C51D (15) mutant proteins also show a single asymmetric resonance around g = 10-12, which tails toward the low field, with broadening into zero field. When analyzed by parallel mode EPR (Fig. 3, dotted lines), the resonances around g = 10-12 from the integer S= 2 [3Fe-4S]^0 clusters are enhanced and sharpened, and the resonances around g = 2 due to the half-integer [4Fe-4S] clusters have disappeared (data not shown).


Figure 3: Perpendicular (solid line) and parallel mode (dotted line) EPR studies of reduced [3Fe-4S] clusters in free PsaC-C14S (A), PsaC-C51S (B), PsaC-C14A (C), and PsaC-C51A (D) mutant proteins. The samples were reduced with sodium dithionite in 330 mM glycine buffer, pH 10.5, containing 0.67% beta-mercaptoethanol. The resonance at 160.5 mT is probably due to a small amount of octahedrally coordinated iron that has remained in the oxidized state. The vertical axis shows signal intensity, with the reduced [3Fe-4S] cluster scaled arbitrarily to unity spin concentration; the comparison depicts relative intensity of the reduced, high spin [4Fe-4S] cluster. Spectrometer conditions were as follows: dual mode resonator, microwave power, 80 mW; microwave frequency, 9.647 GHz (perpendicular mode), 9.349 GHz (parallel mode); modulation amplitude, 10 G at 100 kHz; temperature, 4.2 K.



Evidence for High Spin [4Fe-4S] Clusters in C14S, C51S, C14A, and C51A

The strong asymmetric resonance at g = 4.3 present in PsaC-C14S and PsaC-C51S (Fig. 3, A and B) probably arises from S= d^5 Fe, which is not completely reduced by dithionite. Just downfield of this peak are a new set of broad resonances around g = 5.5, which are difficult to microwave-saturate. This rapidly relaxing spin system is attributed to the low field peak(s) of one or two of the Kramers doublets derived from an S= [4Fe-4S] cluster. The E/D values of high spin [4Fe-4S] clusters are likely to be close to the rhombic limit, with the consequence that the g anisotropy is distributed over 3000 G; hence, the derivative of the low field turning point becomes a broad resonance in which a high concentration of cluster appears as a relatively small peak. Assuming an E/D value of , the midfield resonances would be obscured by other resonances at g = 2, and the highfield resonances are difficult to observe(16) . It is proposed that the resonance(s) around g = 5.5 arise from a high spin, mixed ligand [4Fe-4S] cluster resident in the modified sites of mutant PsaC-C14S and PsaC-C51S. Charged and polar side groups represented by PsaC-C14D(15) , PsaC-C14D (accompanying paper), PsaC-C14S, and PsaC-C51S therefore appear capable of supporting a high spin [4Fe-4S] cluster.

This signal is also present, albeit at a reduced intensity, when the altered amino acid contains a hydrophobic side group such as alanine in PsaC-C14A and PsaC-C51A (Fig. 3, C and D). The intensity of the g 5.5 resonances are considerably weaker in the alanine mutants, but the g value, line shape, and temperature dependence are similar to the aspartate and serine mutants. The implication is that a ligand has been recruited from other than the replacement amino acid to occupy the fourth coordination site of the iron, a good candidate being water, hydroxide, or the free thiolate from beta-mercaptoethanol present in the reconstitution mixture. The occurrence of high spin [4Fe-4S] clusters in the free PsaC proteins is relevant to the finding (see below) that mutant proteins that are rebound to the P700-F(X) core contain two [4Fe-4S] clusters.

Reduction Potentials of the [3Fe-4S] and[4Fe-4S] Clusters in C14S, C51S, C14A, and C51A

To further characterize the properties of the mutant proteins, a redox titration was performed on the [4Fe-4S] clusters of free wild-type PsaC and on the [3Fe-4S] and [4Fe-4S] clusters of free PsaC-C14S, PsaC-C14A, PsaC-C51S, and PsaC-C51A (Table 1). Wild-type PsaC did not titrate with simple Nernstian behavior when the peak at 337 mT and the trough at 360 mT (see Fig. 5) were measured (the features around g = 2.002 were obscured by the redox mediators). Although the resonances became visible at -480 mV and leveled off at -615 mV, the overall shape of the EPR resonances showed subtle differences during the course of the titration, and the n value was a non-integer (data not shown). The titration data could be intepreted to result from two clusters of slightly different potential: one near -500 mV and one slightly more reducing but still less than -600 mV. Alternatively, if the data were forced to a one-component fit, then the midpoint potential of free Synechococcus sp. PCC 7002 PsaC would be about -505 mV, similar to the -510 mV value reported for free spinach PsaC by Hanley et al.(17) . The analysis of the data is complicated by the fact that the line widths are broad, the redox potentials of F(A) and F(B) are reported to be within 60 mV of one another, and the two iron-sulfur clusters are interacting magnetically when both are reduced in the same protein. These factors conspire here to prevent an accurate estimate of the reduction potential of F(A) and F(B) in wild-type PsaC.




Figure 5: EPR spectrum of reduced [4Fe-4S] clusters in free PsaC-C34S, wild-type PsaC, and PsaC-C34A. The clusters were reduced by the addition of a minimal amount of sodium dithionite to a solution of free protein at 2.5 mg/ml in 250 mM glycine, pH 10.5. The g values are not identified due to the broad line widths of the resonances. The magnetic fields differ in the two spectra due to the use of the standard and dual mode resonators; the g values of the principal features of the resonances are nearly equal. Spectrometer conditions were as follows: standard mode resonator; microwave power, 10 mW; microwave frequency, 9.456 GHz; modulation amplitude, 10 G at 100 kHz (A); dual mode resonator; microwave power, 10 mW; microwave frequency, 9.647 GHz; modulation amplitude, 10 G at 100 kHz (B); dual mode resonator; microwave power, 10 mW; microwave frequency, 9.646 GHz; modulation amplitude, 10 G at 100 kHz (C).



The redox titration of the mutant proteins does not have this complication, since the [3Fe-4S] cluster is EPR-visible in the g = 2 region only when oxidized, and the [4Fe-4S] cluster is EPR-visible in the g = 2 region only when reduced. As shown in Fig. 4, A and C, the [4Fe-4S] clusters in the unmodified sites of PsaC-C51S and PsaC-C14S titrate according to a well-behaved Nernstian response with midpoint potentials of -495 mV (n = 1.0) and -520 mV (n = 0.98), respectively. The clusters in the unmodified sites have been driven alternately more oxidizing and more reducing than the comparable clusters in PsaC-C51D (E(m) = -580 mV; (6) ) and PsaC-C14D (E(m) = -515 mV; (6) ). As shown in Fig. 4, B and D, the [3Fe-4S] clusters in the modified sites of PsaC-C51S and PsaC-C14S titrate with midpoint potentials of -145 mV (n = 1.0) and -132 mV (n = 1.0), respectively. The replacement serine drives the potential of the [3Fe-4S] clusters in the modified sites more reducing than the comparable clusters in the PsaC-C51D and PsaC-C14D proteins (E(m) = -98 mV; (6) ). Thus, in the serine series of mutants, the relative potentials of the [4Fe-4S] clusters are inverted, making the serine substitutions of particular interest for functional studies should this inversion of potential be maintained when the mutant proteins are rebound to P700-F(X) cores.


Figure 4: Redox titration of the [4Fe-4S] (panels A and C) and [3Fe-4S] clusters (panels B and D) in free PsaC-C51A (A and B, open squares), PsaC-C51S (A and B, filled circles), PsaC-C14A (C and D, open squares), and PsaC-C14S (C and D, closed circles) mutant proteins. The titrations were carried out by electrochemical poising at pH 9.0. The end points of the titration were determined in the presence of excess potassium ferricyanide and sodium dithionite at pH 10.5 (not shown). The precision of the measurement is shown in the depiction of C51S, which was performed on three independent samples reconstituted at three different occasions. The individually determined values are E(m) = -493, n = 1.01 (squares); E(m) = -493 mV, n = 0.97 (circles); E(m) = -491 mV, n = 1.02 (triangles). Spectrometer conditions were as follows: standard mode resonator, microwave power, 10 mW for the [4Fe-4S] clusters, 1 mW for the [3Fe-4S] clusters; microwave frequency, 9.455 GHz; modulation amplitude, 10 G at 100 kHz; temperature, 15 K for the [4Fe-4S] clusters and 35 K for the [3Fe-4S] clusters.



The substitution of alanine for cysteine was also studied, with the expectation that a hydrophobic amino acid would also affect midpoint potential of the iron-sulfur clusters. As shown in Fig. 4, A and C, the [4Fe-4S] clusters in PsaC-C14A and PsaC-C51A titrate with midpoint potentials of -551 mV (n = 0.98) and -575 mV (n = 0.94), respectively. As shown in Fig. 4, B and D, [3Fe-4S] clusters in PsaC-C51A and PsaC-C14A titrate with midpoint potentials of -167 mV (n = 0.96) and -171 mV (n = 1.04). Thus, in the alanine series of mutants, the midpoint potentials of the [3Fe-4S] clusters are more reducing than in the aspartate and serine mutants. The midpoint potentials of the [4Fe-4S] clusters in the unmodified sites of PsaC-C14D (E(m) = -515 mV) and PsaC-C51D (E(m) = -580 mV) proteins were previously shown to be similar to F(A) and F(B) in wild-type PsaC (Table 1).

Substitution of a Cysteine Not Suspected to Participate in Ligand Binding: EPR Spectra of Two Low Spin [4Fe-4S] Clusters in C34S and C34A

The backbone of PsaC can be modeled to overlay the backbone of P. aerogenes ferredoxin when additional amino acids are inserted in the loop region and added to the C terminus(19, 20) . Cysteine 34 is located in the loop region but at a position that should not allow it to ligate an iron (see Fig. 1). To disallow iron ligation, a protein was constructed in which cysteine 34 was replaced by serine (PsaC-C34S). A conservative replacement amino acid was initially selected to minimize changes to the structure of the protein and to retain similar polarity should this residue be surface-located. The drawback is that serine has been shown to support iron-sulfur clusters in both [2Fe-2S] and [4Fe-4S] proteins. Based on precedent, were PsaC-C34S to ligate an iron of a [4Fe-4S] cluster in PsaC, one would expect to find either a [3Fe-4S] cluster, as shown with PsaC-C14D and PsaC-C51D (6) and with DMSO reductase(21, 22) ; a [4Fe-4S] cluster retained but with altered EPR spectral properties, as shown with the mixed ligand [4Fe-4S] F(X) cluster of PS I(23, 24) ; or a [4Fe-4S] cluster missing, as shown with fumarate reductase (25) .

When the iron-sulfur clusters were inserted into the purified PsaC-C34S apoprotein, the EPR spectrum showed no detectable [3Fe-4S] clusters under mildly oxidizing conditions (not shown). When the PsaC-C34S protein was reduced with sodium dithionite at pH 10.5 (Fig. 5A), the EPR spectrum showed a rhombic signal with broad line widths and with the characteristic splitting observed in proteins that contain two [4Fe-4S] clusters(26) . Indeed, the g values and line widths are nearly identical to those of the [4Fe-4S] clusters representing F(A) and F(B) in free PsaC (Fig. 5B). The clusters in PsaC-C34S and in PsaC have similar but not quite identical relaxation properties, as depicted by the a slight change in line shape as the temperature is lowered from 30 to 5 K (data not shown). Cysteine 34 was also replaced with alanine (PsaC-C34A), an amino acid incapable of providing a ligand to an iron. The EPR spectrum of the holoprotein showed no [3Fe-4S] clusters in the oxidized state, but a rhombic spectrum in the reduced state. As with PsaC-C34S, the g values and line widths are nearly identical to those of the [4Fe-4S] clusters representing F(A) and F(B) in free PsaC (Fig. 5C). The EPR spectroscopic properties of the free PsaC-C34S and the PsaC-C34A proteins are consistent with the proposal that cysteine 34 does not ligate an iron. This assessment is supported by the wild-type EPR spectral properties of F(A) and F(B) in Photosystem I complexes reconstituted with PsaC-C34S and PsaC-C34A (see below).

Simultaneous Substitutions in the Second Cysteine of Both CXXCXXCXXXCP Motifs: Characterization of [3Fe-4S] and [4Fe-4S] clusters in C14D/C51D and C14D/C51D/C34S

A less likely explanation for the high spin [4Fe-4S] clusters in PsaC-C14X and PsaC-C51X (where X represents D, A, and S) is that they arise from the [4Fe-4S] clusters in the unmodified sites through changes induced by a non-cysteine amino acid in the modified sites. While it would be straightforward to address this issue were it possible to accurately count spins, this can be problematic in a protein that contains a mixture of high spin, low spin, and integer spin systems. In principle, it should be possible to oxidize chemically all of the [3Fe-4S] clusters in the modified site, to reduce chemically all of the [4Fe-4S] clusters in the unmodified site, and then to spin count the two S= systems. However, given that the distance betweeen F(A) and F(B) is only 12 Å(27) , there is a further complication that the reduced [3Fe-4S] and [4Fe-4S] clusters may interact magnetically. An alternative approach is to generate a double mutant in which Cys-14 and Cys-51 are both changed to aspartates, PsaC-C14D/C51D. The premise put to the test is that a 2[4Fe-4S] ferredoxin such as PsaC is refolded only when it contains at least one cubane cluster (the naive expectation is that this change would result in a protein that contains two [3Fe-4S] clusters). If the former expectation is borne out experimentally, that the double mutant would contain one or two [4Fe-4S] clusters, then there would be evidence that a mixed ligand site composed of the 3Cysbullet1Asp motif in PsaC is able to support a cubane.

Fig. 6A shows the EPR spectrum of PsaC-C14D/C51D in the presence of sodium ascorbate. The presence of a peak at g = 2.02 and a trough at g = 1.98 indicates the presence of oxidized [3Fe-4S] clusters. When the sample is reduced with dithionite at pH 10.5, a rhombic EPR signal is observed with broad line widths in the g = 2 region that is virtually identical to the low spin [4Fe-4S] cluster seen in the single mutants. This could indicate the presence of one type of cluster located in the F(A) site and another type of cluster located in the F(B) site, or alternatively there may be no site differentiation. Independent of the issue of site distribution of the two types of clusters, a mixed ligand 3Cysbullet1Asp site in PsaC is clearly capable of supporting a cubane. The only distinction is that the spin state of the [4Fe-4S] cluster in the double mutant is S= , while the spin state of the mixed ligand [4Fe-4S] clusters in the single mutants is S= . The likelihood is that the double mutant cannot support two [3Fe-4S] clusters; the implication is that the minimum requirement for a stable protein is the presence in PsaC of at least one cubane cluster.


Figure 6: EPR spectra of the oxidized [3Fe-4S] (dotted line) and reduced [4Fe-4S] (solid line) clusters in free PsaC-C14D/C51D (A), PsaC-C14D/C51D/C34S (B), PsaC-C21D (C), and PsaC-C58D (D) mutant proteins. The proteins were oxidized by brief exposure to air at pH 8.3 and reduced in the presence of sodium dithionite at pH 10.5. The vertical axis shows signal intensity, with the reduced [4Fe-4S] cluster scaled arbitrarily to unity spin concentration; no attempt was made to scale the spin concentration of the [3Fe-4S] clusters relative to the [4Fe-4S] clusters. Spectrometer conditions were as follows: standard mode resonator, microwave power, 10 mW for the [4Fe-4S] clusters, 1 mW for the [3Fe-4S] clusters; microwave frequency, 9.458 GHz; modulation amplitude, 10 G at 100 kHz; temperature, 15 K for the [4Fe-4S] clusters and 35 K for the [3Fe-4S] clusters.



One potential complication is that the nonligating cysteine in position 34 may have been recruited to function as the ligand to one of the two iron-sulfur clusters in the PsaC-C14D/C51D double mutant. A ligand rearrangement has been reported to occur in A. vinelandii ferredoxin I (AvFdI), where C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free cysteine 24 as a replacement ligand(28) . A triple mutant of PsaC was therefore constructed to additionally substitute cysteine 34 with serine: PsaC-C14D/C51D/C34S. By invoking reasoning similar to that for the PsaC-C34S single mutant (see above), were this serine to be recruited to provide a ligand to an iron, the g values and line widths of the [4Fe-4S] cluster should be sufficiently different to distinguish it from the wild type. As shown in Fig. 6B, the EPR spectrum of the triple mutant in the presence of sodium ascorbate shows the presence of a [3Fe-4S] cluster and, after the addition of sodium dithionite at pH 10.5, the presence of an S= , [4Fe-4S] cluster. There are no significant differences in the g values, line shapes, temperature optima, or half-saturation parameters between the double and triple mutants (data not shown). These data verify that both high and low spin [4Fe-4S] clusters can be supported in a mixed ligand 3Cysbullet1Asp environment in PsaC.

Substitutions in the Fourth Cysteine of the CXXCXXCXXXCP Motif: Characterization of [3Fe-4S] and [4Fe-4S] Clusters in C21D and C58D

There exist to our knowledge no naturally occurring or site-directed mutants of ferredoxins with non-cysteine ligands in the proline-proximal position of the CXXCXXCXXXCP iron-sulfur binding motif. If the analogy between PsaC and the ferredoxin of P. aerogenes holds, cysteines 58 and 21 should be located in the interior of PsaC (see Fig. 1), and their size and charge may be crucial in maintaining the three-dimensional structure. Alternatively, the fourth cysteine in each iron-sulfur binding motif also may not be as rigidly constrained by the presence of two nearby, ligating cysteines in the CXXCXXC pattern, is the case in PsaC-C14D and PsaC-C51D. It was therefore of interest to determine whether a substitution of aspartic acid in this position would support the presence of a [3Fe-4S] cluster in this site.

The EPR spectra of the PsaC-C21D and PsaC-C58D mutant proteins are shown in Fig. 6, C and D. In the presence of sodium ascorbate, both proteins show a peak at g = 2.02 and a trough at g = 1.998, characteristic of an oxidized [3Fe-4S] cluster. When PsaC-C21D and PsaC-C58D were reduced with sodium dithionite at pH 10.5, the [3Fe-4S] clusters disappeared and a rhombic EPR signal with broad line widths appeared, which is characteristic of a reduced [4Fe-4S] cluster. The maximum signal intensity of the [4Fe-4S] clusters in PsaC-C21D and PsaC-C58D was achieved at 15 K when measured at 10 mW of power, and the half-saturation parameter (P) was also similar to [4Fe-4S] clusters in the other mutant proteins. These results indicate that it is possible to create a ferredoxin containing a [3Fe-4S] cluster by replacing the fourth as well as the second cysteine in each of the two CXXCXXCXXXCP motifs. A corollary to this finding is that in addition to cysteines 14 and 51, cysteines 21 and 58 also provide ligands to the cubane clusters in PsaC. Since neither PsaC-C21D nor PsaC-C58D rebind to the P700-F(X) core under conditions appropriate for PsaC (shown in Fig. 7), the spectral and redox properties of these proteins were not studied further.


Figure 7: Spectroscopic evidence for the rebinding of PsaC-C34S and PsaC-C34A to P700-F(X) cores. The mutant PsaC proteins were rebound to P700-F(X) cores in the presence of excess PsaD for 12 h and purified as described under ``Materials and Methods.'' For optical studies (A), the solution contained 15 µg/ml chlorophyll, 30 µM dichlorophenol-indophenol, and 0.1 mM sodium ascorbate in 50 mM Tris-HCl, pH 8.3. The onset of the flash is depicted by the arrow; this is followed by a 10-µs instrumental dead time due to the lifetime of the xenon flash and then by the decay of the P700 radical. The measurements were made at 698 nm as described under ``Materials and Methods.'' For EPR studies, the samples were concentrated to 500 µg/ml chlorophyll, 100 µM dichlorophenol-indophenol, and 1 mM sodium ascorbate in 50 mM Tris-HCl, pH 8.3. The spectrum was recorded after freezing the reconstituted PsaC-C34S-PS I complex (B) and the reconstituted PsaC-C34A-PS I complex (C) during illumination. Spectrometer conditions were as follows: standard resonator in panel B at microwave frequency of 9.444 GHz; dual mode resonator in panel C at microwave frequency of 9.645 GHz; microwave power, 20 mW; modulation amplitude, 10 G at 100 kHz, temperature, 15 K. The resonances occur at different field positions in panels B and C due to the resonant frequencies of the standard mode and dual mode cavities.



Rebinding of Mutant PsaC Proteins to P700-F(X)Cores

Fig. 7A shows the time course of the reduction of P700 after a saturating flash in PS I complexes after attempted reconstitution of P700-F(X) cores with PsaC-C34S, PsaC-C34A, PsaC-C21D, PsaC-C58D, and the double mutant PsaC-C14D/C51D. Under conditions appropriate for PsaC rebinding, only PsaC-C34S and PsaC-C34A were effective in restoring the 30-ms back reaction from [F(A)/F(B)]; the other mutants retained the 1.2-ms back reaction from F(X). The EPR spectra of PsaC-C34S-PS I and PsaC-C34A-PS I showed resonances primarily of F(A) when frozen in darkness and illuminated at 15 K (data not shown) and an interaction-type spectrum similar to the wild-type control when frozen under continuous illumination. The close similarity in the g values and line widths of F(A) and F(B) in the PsaC-C34S-PS I complex (Fig. 7B) and the PsaC-C34A-PS I complex (Fig. 7C) with the wild-type PsaC-reconstituted PS I complex (see (7) ) further supports the proposal that this cysteine does not participate in providing a ligand to an iron in PsaC. The analogy with P. aerogenes ferredoxin in terms of cysteine participation in ligand binding thus appears warranted.

When PsaC-C14A, PsaC-C14S, PsaC-C51A, and PsaC-C51S were combined with P700-F(X) cores and analyzed by room temperature optical kinetic spectroscopy, all four mutant reaction centers supported long lived charge separation to F(A)/F(B). Similarly, all reconstituted PS I complexes showed electron transfer to (the equivalent of) F(A) and F(B) when analyzed by low temperature EPR spectroscopy. The detailed electron transfer properties of these mutant reaction center complexes will be reported elsewhere. (^2)


DISCUSSION

The EPR spectroscopic properties of the iron-sulfur clusters in free and PS I-rebound PsaC proteins were determined after the introduction of charged (aspartate), polar (serine), and hydrophobic (alanine) amino acids in the second position and after the introduction of a charged (aspartate) amino acid in the fourth position, of each CXXCXXCXXXCP motif (c.f.Fig. 1). The following generalizations can be made when the second cysteine of the motif is changed. First, a [3Fe-4S] cluster can be found in the oxidized protein and is assumed to be resident in the modified site. The midpoint potentials of the [3Fe-4S] clusters are 400 mV more oxidizing than the wild-type [4Fe-4S] clusters, and the spin relaxation properties, inferred from the temperature dependence and half-saturation parameter, show differences among the three classes of amino acids. Second, a low spin [4Fe-4S] cluster is found in the reduced protein and is assumed resident in the unmodified site. The replacement amino acids, which include those with charged side groups, polar groups, and a hydrophobic side group, show no consistent pattern in affecting the reduction potential of the cluster in the unmodified site (Table 1). Third, high spin [4Fe-4S] clusters are tentatively identified in the PsaC-C14S, PsaC-C51S, PsaC-C14A, and PsaC-C51A reduced proteins and are assumed resident in the modified site. High spin [4Fe-4S] clusters have also been tentatively identified in free C14D-PsaC (15) and free C51D-PsaC (see accompanying paper(36) ).

One likely origin of the high spin (S = ) [4Fe-4S] cluster is the conversion of a reduced [3Fe-4S] cluster as described previously in Peptococcus furiosus(3) and Desulfovibrio africanus ferredoxins(4) , which contain similar CXXDXXCXXXCP motifs. Alternately, the [3Fe-4S] clusters may be derived from the loss of an iron from an oxidized [4Fe-4S] cluster, which may have been inserted in vitro as an intact cubane into the mixed-ligand site of the free PsaC protein. These are the limiting cases; there may well exist a dynamic equilibrium between the [3Fe-4S] and [4Fe-4S] clusters through insertion and loss of an iron controlled by redox potential. While the presence of a high spin [4Fe-4S] cluster was not surprising in the aspartate (or serine) mutants, it was not expected in the alanine mutants. The issue is whether the aspartate and/or serine oxygens provide ligands to the cubane iron as does aspartate in P. furiosus(29, 30) , or whether water, hydroxide, or the thiolate from beta-mercaptoethanol present in the reconstitution mixture, has contributed the fourth ligand. In this respect, it is interesting that only those mutant proteins that show evidence for a high spin [4Fe-4S] cluster in the mutant site are capable of reconstituting onto P700-F(X) cores.

An additional new finding is that the PsaC-C14D/C51D double mutant does not contain two [3Fe-4S] clusters; rather it contains [3Fe-4S] and low spin [4Fe-4S] clusters in a nearly stoichiometric ratio (data not shown). In light of the above data on the single mutants, it is intriguing that two [4Fe-4S] clusters are not present in the PsaC-C14D/C51D double mutant. The presence of both types of clusters in the PsaC-C14D/C51D double mutant is in agreement with the premise that PsaC must contain at least one cubane cluster for stability. It is also interesting that an earlier attempt to introduce two [3Fe-4S] clusters into A. vinelandii Fd I also failed(28) . This protein normally contains one [3Fe-4S] and one [4Fe-4S] cluster. When a cysteine ligand of the [4Fe-4S] cluster was altered, a nearby cysteine was recruited as a replacement ligand, resulting in a protein with structural modifications but still containing one [3Fe-4S] and one [4Fe-4S] cluster. In an attempt to minimize the chance that cysteine 34 provides a ligand to one of the clusters in PsaC-C14D/C51D, the triple mutant PsaC-C14D/C51D/C34S was constructed. This mutant does not contain a recruitable cysteine residue; hence, the [4Fe-4S] cluster must reside in a mixed ligand site. Nevertheless, serine cannot be dismissed as a potential ligand since it supports a [2Fe-2S] cluster with modified optical and EPR properties in a site-directed mutant of ferredoxin (31, 32, 33) and NADH-quinone oxidoreductase (34) and a low spin [4Fe-4S] cluster with modified EPR properties in a site-directed mutant of the interpolypeptide F(X) cluster in PS I(23, 24) . However, the g values, line widths, temperature optimum, and P values of the PsaC-C14D/C51D/C34S mutant were identical to those of the PsaC-C14D/C51D double mutant, in agreement with the proposal that cysteine 34 is not a ligand to a cubane iron in this instance.

The detection of a high spin [4Fe-4S] cluster in the C14X and C51X (where X represents A, D, or S) single mutants provides a precedent for the presence of a [4Fe-4S] cluster in the C14D/C51D double mutant. The only significant difference is the S = spin state of the [4Fe-4S] cluster in the single mutants and the S = spin state of the [4Fe-4S] cluster in the double mutant. It should be noted that P. furiosus ferredoxin contains a single, mixed ligand [4Fe-4S] cluster that exists in both S = (20%) and S = (80%) ground states(3) . The cross-over energy between the high and low spin states is likely to be small; indeed, ethylene glycol and urea change the spin state in the A. vinelandii ferredoxin(35) . One factor appears to be related to the degree of exposure to solvent, a consideration that may be relevant in the spin state changes that have been postulated to occur between the free and PS I-rebound PsaC-C51D(15) .

In summary, these studies indicate that PsaC refolds only in the presence of two iron-sulfur clusters when cysteine is replaced in positions 14 and 51 by aspartate, serine, and alanine, and in positions 21 and 58 by aspartate. Since there was no occurrence of a mutant PsaC that contained only one [3Fe-4S] cluster, only one [4Fe-4S] cluster, or two [3Fe-4S] clusters, a corollary to the above premise is that a stable three-dimensional structure may require that two conditions be met: 1) two iron-sulfur clusters are present, and 2) one of the two clusters is a cubane. The inability to observe a [3Fe-4S] cluster in the C14D/C51D-PS I complex implies that it may be difficult to introduce this type of motif into PsaC in vivo. In light of photosynthesis, two points can be made. First, the important feature for electron transfer is that the mixed ligand, [4Fe-4S] clusters in reconstituted PsaC-C14D-PS I(34) , PsaC-C14S-PS I, PsaC-C14A-PS I, PsaC-C51D-PS I(15) , PsaC-C51S-PS I, and PsaC-C51A-PS I complexes are capable of accepting electrons at both cryogenic and room temperatures. Second, a checkpoint for the assembly of PsaC onto PS I cores may be that the free PsaC protein must contain two [4Fe-4S] clusters.


FOOTNOTES

*
This research was supported by National Science Foundation Grants MCB-9205756 (to J. H. G.) and MCB-9206851 (to D. A. B.). Published as Journal Series No. 11064 of the University of Nebraska Agricultural Research Division. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Biotechnology Career Fellowship from the Rockefeller Foundation.

Supported by Cooperative Agreement from the National Science Foundation/EPSCoR (OSR-9255225).

**
To whom correspondence should be addressed. Tel.: 402-472-2931; Fax: 402-472-7842; jgolbeck@unlinfo.unl.edu.

(^1)
The abbreviations used are: PS I, photosystem I; EPR, electron paramagnetic resonance; E(m), midpoint potential (versus hydrogen); MES, 4-morpholineethanesulfonic acid; mW, milliwatt(s); mT, milliteslas.

(^2)
Jung, Y-S., Vassiliev, I., Qiao, F., Bryant, D., and Golbeck, J. H., manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Gautam Sarath for performing the protein sequence determinations and Dr. Michael Johnson for valuable discussions on the properties of high spin [4Fe-4S] clusters.


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