Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry*

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

In photosynthetic electron transfer, photosystem I (PSI) 2 functions as an integral light-driven plastocyanin:ferredoxin oxidoreductase. It is a thylakoid-embedded multiprotein complex that consists of ϳ14 subunits in Chlamydomonas reinhardtii (1). Using light energy, PSI transports electrons from the lumenal soluble electron carrier plastocyanin (pc) across the membrane to the stromal soluble electron acceptor ferredoxin. Under copper deficiency, pc can be functionally replaced in green algae and in cyanobacteria by cytochrome c 6 (cyt c 6 ) (2)(3)(4)(5).
Electron transfer between pc or cyt c 6 and the eukaryotic PSI can be described by a multistep model of donor binding, PSIdonor complex formation, electron transfer, and donor unbinding (6,7). In eukaryotic organisms, binding of pc or cyt c 6 to the PSI is mainly driven by two different forces, electrostatic attraction and hydrophobic contact. Long range electrostatic attractions between basic patches of PsaF and acidic regions of pc and cyt c 6 (8 -12) as well as the hydrophobic contact between the electron transfer site of the donors and PSI, including PsaA-Trp 651 and PsaB-Trp 627 in C. reinhardtii (9,13), are required for stable electron transfer complex formation and efficient electron transfer. Cross-linking studies, knock-out, and reverse genetics experiments have established the crucial function of the positively charged eukaryotic N-terminal domain of PsaF for the binding of both donors (10, 12, 14 -16). Hereby, Lys 23 of PsaF from C. reinhardtii was found to be the key residue for the electrostatic interaction between PSI and both donors (12). The positively charged N-terminal domain is missing in prokaryotic organisms, and correspondingly, knock-out experiments and functional electron transfer measurements have demonstrated that, in the cyanobacterium Synechocystis sp. PCC6803, efficient binding and electron transfer between PSI and pc or cyt c 6 are independent of PsaF (10,17,18). Introduction of the basic PsaF domain from C. reinhardtii into cyanobacterial PsaF allowed efficient binding and fast electron transfer between the algal donors and the chimeric cyanobacterial PSI, proving that the positively charged N-terminal domain of PsaF is essential for electrostatic attraction of the donors and formation for the electron transfer complex competent in fast electron transfer (15).
As outlined, in cyanobacteria, electrostatic interactions between the electron donors and the oxidizing side of PSI are independent of PsaF or any other peripheral PSI subunit. However, site-directed mutagenesis of pc or cyt c 6 from Synechocystis sp. PCC 6803 and Anabaena PCC 7119 reveal the importance of electrostatic attractions between the reaction partners. Analysis of binding and electron transfer between the altered donors and the cyanobacterial PSI revealed that a positively charged amino acid located at the "northern face" of either pc or cyt c 6 is crucial for the interaction with the reaction center (19 -21). Interestingly, an equivalent positively charged exposed amino acid is present in cyt c 6 from C. reinhardtii (22) but absent from pc of C. reinhardtii (23) and other eukaryotic pc structures. It is tempting to speculate that this positively charged amino acid expressed by pc or cyt c 6 in cyanobacteria is important for electrostatic recognition of a negatively charged amino acid present in the core subunits PsaA or PsaB, thereby helping the formation of an electron transfer complex.
It is of note that the hydrophobic interaction site of the PSI core formed by PsaA and PsaB with the electron transfer donors is conserved between prokaryotic and eukaryotic organisms. The high resolution structural data of cyanobacterial and pea PSI (24,25) show that a Trp residue (corresponding to Trp 627 in C. reinhardtii) from a luminal helix of PsaB together with a Trp residue (corresponding to Trp 651 in C. reinhardtii) from a luminal helix of PsaA form a sandwich-like structure directly situated above P700. The importance of the lumenal loop j of the PsaB for electron transfer between PSI and the soluble donors has been demonstrated in Synechocystis PCC 6803. A double mutation in the lumenal loop j of the PsaB (W622C/A623R) caused a highly photosensitive phenotype and exhibited a severe defect in the interaction and electron transfer with pc or cyt c 6 (26). The function of the two Trp residues in electron transfer between the two donors and PSI was directly analyzed in a reverse genetic experiment in C. reinhardtii. Alteration of the Trp residues by site-directed mutagenesis and transformation of PsaA-and PsaB-deficient strains resulted in the generation of PsaA and PsaB mutant strains PsaA-Trp651Phe, PsaA-Trp651Ser, and PsaB-Trp627Phe (corresponding to PsaA-Trp 655 PsaB-Trp 631 in Synechococcus) (13,27). The change of either of the two PsaA or PsaB Trp residues to Phe abolished the formation of an intermolecular electron transfer complex between the altered PSI and pc, indicating that PsaATrp 651 as well as PsaBTrp 627 of loops i/j form the hydrophobic recognition site required for binding of pc to the core of PSI.
In respect to electrostatic and hydrophobic recognition sites of the eukaryotic donor molecules, only pc has been investigated in detail. Site-directed mutagenesis of eukaryotic pc has demonstrated that the "southern" conserved negatively charged patch and the northern hydrophobic flat site of pc are required for electrostatic attraction and hydrophobic contact between pc and PSI, respectively, to promote binding and efficient electron transfer between pc and PSI (8 -10,28).
The precise electrostatic recognition site of eukaryotic cyt c 6 that enables specific binding to the positively charged domain of PsaF is unknown. To identify the amino acid on cyt c 6 responsible for binding to PsaF, we isolated a chemically crosslinked complex between cyt c 6 and PSI from C. reinhardtii that was competent in fast and efficient electron transfer. The active cross-linked complex was digested with trypsin, and peptides were analyzed by mass spectrometry. The mass spectrometric analysis identified cross-linked peptides and amino acids on cyt c 6 and PsaF that, in turn, allows defining the precise recognition sites on both proteins. Using the crystal structures for eukaryotic PSI (25) and eukaryotic cyt c 6 (22) and the genetic and biochemical data ( (12,13,27) and this study), we present and discuss a model of the intermolecular complex between the two electron transfer partners.

MATERIALS AND METHODS
Strains and Media-C. reinhardtii wild-type strain was grown as described in Tris acetate phosphate medium with or without copper (29).
Isolation of pc and Cyt c 6 -The isolation of pc and cyt c 6 followed published procedures (4, 11). The concentrations of pc and cyt c 6 were determined spectroscopically using an extinction coefficient of 4.9 mM Ϫ1 cm Ϫ1 at 597 nm for the oxidized form of pc (30) and 20 mM Ϫ1 cm Ϫ1 at 552 nm for the reduced form of cyt c 6 (2). Samples were tested for contamination of each other by immunodetection using anti-pc or anticyt c 6 antibodies.
Isolation of Thylakoid Membrane PSI Complex-The isolation of thylakoid membranes purified by centrifugation through a sucrose step gradient and the isolation of PSI particles was as described previously (11). Chlorophyll concentrations were determined according to Porra (31).
SDS-PAGE (15.5% T, 2.66% C; % T is the polyacrylamide concentration at a constant concentration of the cross-linking agent bisacrylamide (% C)) was carried out according to Laemmli (32). After the electrophoretic fractionation, the protein bands were visualized by Coomassie staining, and bands corresponding to the PsaF-cyt c 6 cross-link product were excised.
Cross-linking Procedure-Cross-linking was performed as described previously (11). Cross-linked particles were fractionated by SDS-PAGE separation, and the cross-linked band was excised and digested with trypsin as described previously (33). For kinetic analysis, the cross-linking reaction was stopped by dilution, and particles were collected as described previously (11).
Flash Absorption Spectroscopy-Kinetics of flash-induced absorbance changes at 817 nm were measured essentially as described previously (6,12). The measuring light was provided by a luminescence diode (Hitachi HE8404SG, 40 milliwatt, full width at half-maximum 30 nm) supplied with a stabilized battery-driven current source. The light was filtered through a 817-nm interference filter (full width at half-maximum 9 nm) and passed through a cuvette containing 200 l of the sample with an optical path-length of 1 cm.
Mass Spectrometry-For mass spectrometric identification of the peptides, the excised protein bands were destained with 70% ethanol, 10% acetic acid, cut in small pieces, and subjected to trypsin digestion as described previously (34). Liquid chromatography-tandem mass spectrometry analysis was performed on a LCQ Deca XP Plus ion trap mass spectrometer (Thermo Electron, San Jose, California) in combination with a Famos autosampler, Switchos valve machine, and an Ultimate high pressure liquid chromatography machine (LC-Packings, Sunnyvale, California) as described previously (34). For reversed phase chromatography, a linear gradient over 90 min with an initial aqueous phase of 0.1% (v/v) formic acid in 5:95 acetonitrile:water and a final organic phase of 0.1% (v/v) formic acid in 80:20 acetonitrile:water was used. Acquisition of mass spectra and sequence identification using Sequest software (Thermo Electron, San Jose, California) were done as described previously, except that the range of peptide masses (MH ϩ ) was 200 -7500. The data base used for the peptide search was constructed of photosynthetic components of Chlamydomonas. To detect cross-linked peptides, Glu or Asp residues were allowed up to six different additional masses per run when searching the spectra against the data base with the Sequest software. The masses corresponded to various tryptic fragments of PsaF, allowing up to two missed cleavages and searching in different combinations of the different additional masses.
Model Construction-The structure of C. reinhardtii cyt c 6 is already solved (Protein Data Bank (PDB) entry 1cyj) (22) and was used for the model presented here. To create a model of the electron transfer complex between PSI and cyt c 6 , the C. reinhardtii PsaA, PsaB, and PsaF sequences were modeled to the eukaryotic PSI structure (PDB entry 1yo9), which is a model derived from the crystal structure of a eukaryotic PSI and a cyanobacterial PSI structure (35). Sequences for each PSI subunit were obtained from the Chlamydomonas genome project version 3.0 and homology models for each subunit were constructed individually using the Swiss model online server (36) and the Swiss PDB viewer (37). The cyt c 6 was then docked by hand to the PSI complex taking the biological data of a distance of ϳ14 Å between the redox cofactors and the data presented here into account. The model was energy-minimized using the Swiss PDB viewer implemented GROMOS96.

RESULTS
Cross-linking between PSI and Cyt c 6 -Cross-linking of PSI with cyt c 6 resulted in a cross-linked product visible as an additional band after SDS-PAGE separation and Coomassie staining of the reaction mixture (Fig. 1). The band appeared in the mass range of the PsaF-cyt c 6 cross-linked product, as described previously, by immunodetection with anti-PsaF antibodies (13). The band was excised, digested with trypsin, and used for further mass spectrometric analysis.

The Cross-linked Complex between PSI and Cyt c 6 Is Competent in Fast Electron Transfer as Revealed by Flash-induced
Absorption Spectroscopy-To investigate the functionality of the cross-linked complex between PSI and cyt c 6 , the isolated cross-linked particles were analyzed by flash-induced absorption spectroscopy. The resulting kinetic traces (Fig. 2) can be deconvoluted by a bi-exponential decay revealing three distinct populations of PSI in the analyzed sample. The fast phase shows the same properties as the intermolecular electron transfer complex reaction between PSI and cyt c 6 , with a half-time of 4 s. The amplitude of this fast phase corresponds to 60% of the total amplitude. Because this kinetic component is extremely sensitive to changes in distance and geometry within the electron transfer complex, we conclude that, in more than half of the cross-linked particles, PSI is cross-linked in a functional way that resembles the in vivo native complex. The second, slower phase with a half-time of 42 s and a minor amplitude of 6.8% in respect to the total amplitude might relate to a crosslink product obeying a distorted geometry of the electron transfer partners. The constant fraction with 33.2% of the total amplitude is reduced in a different timescale and corresponds to not cross-linked or not functionally cross-linked PSI, which is reduced by ascorbate.
Mass Spectrometric Analysis Reveals Specific Sites of Protein-Protein Interaction-Mass spectrometric analysis of the excised and tryptic digested protein band revealed PsaF and cyt c 6 as expected. The identified peptide sequences and recovery rates are summarized in Table 1.
For the identification of cross-linked peptides using Sequest, Glu and Asp residues were allowed additional masses that corresponded to tryptic peptides of PsaF, allowing up to two missed cleavages (minus H 2 O for the cross-link peptide bound). The rationale behind this is that we expected to find cyt c 6 peptides that were cross-linked via Lys residues of PsaF. Because the Sequest software allows only up to six modifications, the mentioned additional masses were searched in different combinations.  In complete digests, we found no significant hits for queries that included PsaF-peptides that contained the C-terminal Lys residue as the only Lys residue. This was expected, because cross-linking the ⑀-amino group of a Lys residue to the ␥/␦carboxyl group of Asp/Glu residue results in loss of the recognition site for peptide hydrolysis by trypsin.
For queries with PsaF peptides that include one missed cleavage site, we found peptides 24 TLEKR 28 and 21 ELKTLEK 27 cross-linked with the cyt c 6 peptide 67 LSEEEIQAVAEYVFK 81 . Allowing two missed cleavage sites, we identified PsaF peptide 20 KELKTLEK 27 cross-linked to the same cyt c 6 peptide (summarized in Table 2). Because of the fact that all PsaF peptides were linked to the same cyt c 6 peptide 67 LSEEEIQA-VAEYVFK 81 and this peptide was also found by Sequest by itself (see Fig. 3A), additional cross-linking/interaction sites between cyt c 6 and the basic patch of PsaF could be predicted. It should be of note that the cross-linking products were also found by Sequest searches when looking for PsaF tryptic peptides modified on Lys with the mass corresponding to the cyt c 6 peptide 67-81 (data not shown), although the Xcorr factors were significantly lower. This demonstrates that peptide length and query identity strongly influences the outcome of the Sequest algorithm. This is not surprising, because these cutoff values were designed for simple linear peptides and have less significance for the more complex cross-linked peptides.
Unfortunately, Sequest analysis did not allow for discrimination of the specific sites of cross-linking (Glu 69 /Glu 70 /Glu 71 on cyt c 6 or Lys 20 /Lys 23 on PsaF for 20 KELKTLEK 27 ) and gave sig-nificant values for the different proposed products. Therefore, we manually examined the tandem mass spectra for discriminating y and b ions. We did not find specific tandem mass spectrometry fragments to discriminate between Glu 69 and Glu 70 for the cross-link with 20 KELKTLEK 27 and 21 ELK-TLEK 27 of PsaF. However, for the peptide PsaLys 20 -Lys 27 , we found the series yЈ 2ϩ 4 -yЈ 2ϩ 7, indicating Lys 23 to be the linking residue. For the PsaF peptide 24 TLEKR 28 crosslinked to the cyt c 6 peptide 67 LSEEEIQAVAEYVFK 81 , we found y 12ϩ , y 2ϩ 12 , b 3ϩ , and b 2ϩ 3 ions indicating a cross-link between Glu 69 and Lys 27 (see Fig. 3C).
As stated above, we expected other cross-linked peptides. Therefore, we carefully re-examined other Sequest hits that were below the significance cutoff and checked their significance with an extended tandem mass spectrometry product table, including y and b ions as well as yЈ and bЈ ions. Indeed we found an additional cross-linking product, cyt c 6 -58 GAM-PAWADR 66 cross-linked to PsaF-13 AYAKLEK 19 , implying a cross-link between Asp 65 of cyt c 6 and Lys 16 of PsaF. The peptide cyt c 6 -58 GAMPAWADR 66 was not found in the tandem mass spectrometry analysis of the total cross-linked peptides. However, this is not related to cross-linking efficiency but rather to a feature of the "flyability" of the peptide, because it was not detected in an tandem mass spectrometry experiment with only purified and trypsin-digested cyt c 6 (data not shown). Looking at the resulting geometry of the PSI and cyt c 6 crosslinked complex, it seems rather unlikely that this product is part of the proper electron transfer complex (see "Discussion"). We

Recovery of peptides from PsaF and cytochrome c 6 by MS
The underlined sequences were found by MS, and the italic sequences were found as cross-linked products. Gly 58 -Arg 66 of cytochrome c 6 and Ala 13 -Arg 28 of PsaF were only found in cross-linked peptides.   The insets show the mass peaks from which the tandem mass spectra were achieved. In B and C, only the y and b peaks unique to the cross-linked peptide are indicated; all other major peaks derive from the peptide fragments, which do not carry the cross-linking site. In C, the peaks indicative for the specific cross-link between Glu 69 (cyt c 6 ) and Lys 27 (PsaF) are in italic.

Recovery
Recognition Sites between Cytochrome c 6 and Photosystem I NOVEMBER 17, 2006 • VOLUME 281 • NUMBER 46 propose that this PSI and cyt c 6 cross-linked complex corresponds to the fraction of PSI with a slower reduction half-time of 42 s and a distorted geometry.

DISCUSSION
To elucidate the electrostatic interaction between cyt c 6 and PSI on the molecular level, we took advantage of chemical cross-linking, which resulted in a zero-length cross-linked complex between PsaF and cyt c 6 . Importantly, the cross-linked complex was competent in fast and efficient electron transfer, indicating that cross-linking conserved the authentic orientation of the electron transfer partners in the complex. Mass spectrometric analyses of tryptic peptides from the crosslinked product between cyt c 6 and PsaF revealed specific interaction sites between residues Lys 27 of PsaF and Glu 69 of cyt c 6 and between Lys 23 of PsaF and Glu 69 /Glu 70 of cyt c 6 . Interestingly, this identifies the Lys residues of PsaF, in particular Lys 23 , which are implicated in binding of pc and cyt c 6 , as demonstrated by the reverse genetics experiments (12). This recognition site is also consistent with pea PSI crystal structure (25). In respect to the soluble electron donor and binding partner, this recognizes Glu 69 /Glu 70 of cyt c 6 as electrostatic partner sites. Glu 69 and Glu 70 are located at the "eastern face" of cyt c 6 . In cyanobacteria, a positively charged amino acid located at the northern face of either pc or cyt c 6 is crucial for the interaction with the reaction center (19 -21). Interestingly, an equivalent positively charged, exposed amino acid is present in cyt c 6 from C. reinhardtii (22). Although the residue on PSI interacting with this positively charged, exposed amino acid is unknown, our result demonstrates that the negatively charged residues Glu 69 /Glu 70 of cyt c 6 bind to Lys 23 and Lys 27 of PsaF and represent the electrostatic recognition site of eukaryotic cyt c 6 .
In Fig. 4, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c 6 and PSI. In this model, the distance between the redox cofactors is ϳ14 Å, as estimated by Hippler et al. (10), and the Glu 69 and Glu 70 of cyt c 6 are able to form a strong salt bridge with Lys 27 and Lys 23 of PsaF, respectively, as the mass spectrometric data indicate. Lys 20 and Lys 16 form weaker salt bridges with Glu 71 of cyt c 6 and Glu 613 of PsaB, respectively. This is in accordance with the finding that Glu 613 of PsaB functions to keep the N-terminal part of PsaF in a proper position to enable an effective electron transfer complex (13). Interestingly, in this model, the mentioned conserved positive charge on the northern face of cyt c 6 (Arg 66 ) and the adjacent Asp 65 can form a strong salt bridge with the pair Arg 623 /Asp 624 of PsaB. This again is in line with the finding that, when disrupting the hydrophobic interaction area for the electron donors on PSI, plastocyanin, which lacks this additional recognition site, is more compromised in complex formation with PSI than cyt c 6 (27).