The role of axial ligands for the structure and function of chlorophylls

We have studied the effect of axial ligation of chlorophyll and bacteriochlorophyll using density functional calculations. Eleven different axial ligands have been considered, including models of histidine, aspartate/glutamate, asparagine/glutamine, serine, tyrosine, methionine, water, the protein backbone, and phosphate. The native chlorophylls, as well as their cation and anion radical states and models of the reaction centres P680 and P700, have been studied and we have compared the geometries, binding energies, reduction potentials, and absorption spectra. Our results clearly show that the chlorophylls strongly prefer to be five-coordinate, in accordance with available crystal structures. The axial ligands decrease the reduction potentials, so they cannot explain the high potential of P680. They also redshift the Q band, but not enough to explain the occurrence of red chlorophylls. However, there is some relation between the axial ligands and their location in the various photosynthetic proteins. In particular, the intrinsic reduction potential of the second molecule in the electron transfer path is always lower than that of the third one, a feature that may prevent back-transfer of the electron.     

Bidirectional electron transfer in photosystem I: accumulation of A0- in A-side or B-side mutants of the axial ligand to chlorophyll A0

Photosystem I contains two potential electron transfer pathways between P(700) and F(X). These branches are made up of the electron transfer chain components A, A(0), and A(1). The primary electron acceptor A(0) is a chlorophyll a monomer that could be one or both of the two chlorophyll molecules, eC-A(3)/eC-B(3), identified in the 2.5 A resolution structure. The eC-A(3)/eC-B(3) chlorophylls are both coordinated by the sulfur atom of a methionine. This coordination is highly unusual, as interactions between the acid Mg(2+) and the soft base sulfur are weak. The eC-A(3)/eC-B(3) chlorophylls also are located close to one of the connecting chlorophylls that may link the antenna and the electron transfer chain chlorophylls. Due to their location in the structure, the eC-A(3)/eC-B(3) chlorophylls may play a role in both excitation energy transfer and electron transfer. To test the role of the eC-A(3)/eC-B(3) chlorophylls in electron transfer, Met-684 of PsaA and Met-664 of PsaB have been changed to His, Ser, and Leu. Replacement of either M(A684) or M(B664) results in a significant alteration in growth phenotype. The His and Leu mutants are very light sensitive in the presence of oxygen. Growth is impaired to a greater extent in the B-side mutants. However, all of the mutants are able to grow anaerobically at comparable rates. The His and Ser mutants all accumulate PSI at a level similar to that of wild type, whereas the Leu mutants have reduced amounts of PSI. Ultrafast transient absorbance measurements show that the (A(0)(-) - A(0)) difference signal accumulates in the MH(A684) and MH(B664) mutants under neutral conditions, demonstrating that electron transfer between A(0)(-) and A(1) is blocked or significantly slowed. The results show that both the A-branch and the B-branch of the ETC are active in PSI from Chlamydomonas reinhardtii.     

Kinetics and spectra of photo-induced changes in the absorption of pigment-protein complexes of photosystem 1 in a picosecond range

The energy transfer from the light-harvesting antenna chlorophylls to the reaction center molecules and subsequent charge separation were investigated using a difference picosecond spectrophotometer with selective excitation. The objects were the pigment-protein complexes of photosystem 1 (Chl/P700 = 60) isolated from bean leaves. The difference absorption spectra of the excited states of light-harvesting antenna chlorophylls and the P700 photooxidation were measured. It was shown that the excited states of antenna chlorophylls were generated within 10 ps and deactivated with three-component kinetics: tau 1 = 20--45 ps, tau 2 = 100--300 ps, tau 3 greater than 500 ps. The process of the P700 photooxidation induced by the 650 nm exciting pulse was approximately monoexponential with tau equal to 15--30 ps. It is established that the P700 photooxidation is due to the efficient transfer of excitation energy from antenna chlorophylls to reaction centers.     

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., K?ss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).     

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.     

Dynamics of excitation in the photosystem I of cyanobacteria: transfer in the antenna, capture by the reaction site, and dissipation

The structure of a complex of photosystem I (PSI) of cyanobacteria and the mechanisms of the functioning of the antenna and PSI reaction site were described. The complex of PSI in thylakoids of cyanobacteia is organized as a trimer whose antenna is enriched in long-wave chlorophylls. The energy absorbed by these chlorophyls migrates to P700, inducing its oxidation. Long-wave chlorophyls are also involved in the dissipation of excessive energy; both the cation radical of P700 and the triplet of P700 effectively quench the fluorescence of long-wave chlorophyll of PSI. The energy exchange between the antennas of monomers in the trimer of PSI stimulates the dissipation of electron excitation energy, protecting the complex against photodestruction. The kinetics of energy migration in the antenna and charge separation in the reaction site of PSI trimers was studied using subpicosecond spectroscopy. Long-wave chlorophylls of PSI do not substantially affect the energy migration in the heterogeneous antenna of PSI but slow down the capture of energy of P700. The separation of changes in the reaction site of PSI is the most rapid among the known reaction sites.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

Excitonic interactions in wild-type and mutant PSI reaction centers

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.     

Subunit composition of Photosystem I complex that catalyzes light-dependent transfer of electrons from plastocyanin to ferredoxin.

The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.     

Ultrafast primary processes in PS I from Synechocystis sp. PCC 6803: roles of P700 and A(0)

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10(-5) absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at approximately 680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700(+) - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 ( approximately 24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 --> A(0) electron transfer is 9-10 ps, whereas the kinetics of the subsequent A(0) --> A(1) electron transfer are still unknown.     

Electron transfer from plastocyanin to the photosystem I reaction center in mutants with increased potential of the primary donor in Chlamydomonas reinhardtii

The dependence of the P(700)(+)/P(700) midpoint potential on kinetics of reduction of P(700)(+) in vivo has been examined in a series of site-directed mutants of Chlamydomonas reinhardtii in which the histidyl axial ligand to the Mg(2+) of the P(700) chlorophyll a has been changed to several different amino acids. In wild-type photosystem I, the potential of P(700)(+)/P(700) is 447 mV and the in vivo half-time of P(700)(+) reduction by its natural donor, plastocyanin, is 4 micros. Substitution of the axial histidine ligand with cysteine increases the potential of P(700)(+)/P(700) to 583 mV and changes the rate of P(700)(+) reduction to 0.8 micros. Mutants with a range of potentials between 447 and 583 mV show a strong correlation of the P(700)(+)/P(700) potential to the rate of reduction of P(700)(+) by plastocyanin. There is also an increase in the rate of photosystem I-mediated electron transfer from the artificial electron donor DCPIP to methyl viologen in thylakoid membranes. The results indicate that the overall rate constant of P(700)(+) reduction is determined by the rate of electron transfer between the copper and P(700)(+) and confirmed that in vivo there is a preformed complex between plastocyanin and photosystem I. Using approximations of the Marcus electron transfer theory, it is possible to estimate that the distance between the copper of plastocyanin and P(700)(+) is approximately 15 A. On the basis of this distance, the plastocyanin docking site should lie in a 10 A hollow formed by the lumenal exposed loops between transmembrane helices i and j of PsaA and PsaB.     

Species-specific differences of the spectroscopic properties of P700: analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii

We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700-P700) and (P700+.-P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700-P700), the (P700+.-P700) absorbance difference and in the (P700+.-P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra.     

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.     

Orientation of Photosystem-I pigments: low temperature linear dichroism spectroscopy of a highly-enriched P700 particle isolated from spinach

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The Q_Y transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700⁺ and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A₀, are all preferentially oriented perpendicular to the gel squeezing direction. The Q_Y transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.Abbreviations PSI Photosystem I - P700 primary electron donor of PSI - A₀ primary electron acceptor of PSI     

A comparative flash-photolysis study of electron transfer from pea and spinach plastocyanins to spinach Photosystem 1. A reaction involving a rate-limiting conformational change

Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.Abbreviations cyt- cytochrome - IPTG- isopropyl--d-thiogalactopyranoside - P,P700- reaction-center chlorophyll - Pc- plastocyanin - PS 1- Photosystem 1 - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - WT- wild-type     

Primary donor photo-oxidation in photosystem I: a re-evaluation of (P700(+) - P700) Fourier transform infrared difference spectra.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the photo-oxidation of the primary electron donor (P700) in PS I particles from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. To aid in the interpretation of the spectra, PS I particles from a site-directed mutant of C. reinhardtii, in which the axial histidine ligand (HisA676) was changed to serine, were also studied. A high-frequency (3300-2600 cm(-1)) electronic transition is observed for all PS I particles, demonstrating that P700 is dimeric. The electronic band is, however, species-dependent, indicating some differences in the electronic structure of P700 and/or P700(+) in C. reinhardtii and Synechocystis sp. 6803. For PS I particles from C. reinhardtii, substitution of HisA676 with serine has little effect on the ester carbonyl modes of the chlorophylls of P700. However, the keto carbonyl modes are considerably altered. Comparison of (P700(+) - P700) FTIR difference spectra obtained using PS I particles from the wild type (WT) and the HS(A676) mutant of C. reinhardtii indicates that the mutation primarily exerts its influence on the P700 ground state. The 13(1) keto carbonyls of the chlorophylls of P700 of the wild type absorb at similar frequencies, which has previously made these transitions difficult to resolve. However, for the HS(A676) mutant, the 13(1) keto carbonyl of chlorophyll a or chlorophyll a' of P700 on PsaB or PsaA absorbs at 1703.4 or 1694.2 cm(-1), respectively, allowing their unambiguous resolution. Upon P700(+) formation, in both PS I particles from C. reinhardtii, the higher-frequency carbonyl band upshifts by approximately 14 cm(-1) while the lower frequency carbonyl downshifts by approximately 10 cm(-1). The similarity in the spectra for WT PS I particles from C. reinhardtii and Synechocystis sp. 6803 indicates that a similar interpretation is probably valid for PS I particles from both species. The mutant results allow for an interpretation of the behavior of the 13(1) keto carbonyls of P700 that is different from previous work [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592], in which it was suggested that 13(1) keto carbonyls of P700 absorb at 1697 and 1639 cm(-1), and upshift by 21 cm(-1) upon cation formation. The interpretation of the spectra reported here is more in line with recent results from ENDOR spectroscopy and high-resolution crystallography.     

Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI

A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.     

Excitation dynamics and heterogeneity of energy equilibration in the core antenna of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Energy equilibration in the photosystem I core antenna from the cyanobacterium Synechocystis sp. PCC 6803 was studied using femtosecond transient absorption spectroscopy at 298 K. The photosystem I core particles were excited at 660, 693, and 710 nm with 150 fs spectrally narrow laser pulses (fwhm = 5 nm). Global analysis revealed three kinetic processes in the core antenna with lifetimes of 250-500 fs, 1.5-2.5 ps, and 20-30 ps. The first two components represent strongly excitation wavelength-dependent energy equilibration processes while the 20-30 ps phase reflects the trapping of energy by the reaction center. Excitation into the blue and red edge of the absorption band induces downhill and uphill energy flows, respectively, between different chlorophyll a spectral forms of the core. Excitation at 660 nm induces a 500 fs downhill equilibration process within the bulk of antenna while the selective excitation of long-wavelength-absorbing chlorophylls at 710 nm results in a 380 fs uphill energy transfer to the chlorophylls absorbing around 695-700 nm, presumably reaction center pigments. The 1.5-2.5 ps phases of downhill and uphill energy transfer are largely equivalent but opposite in direction, indicating energy equilibration between bulk antenna chlorophylls at 685 nm and spectral forms absorbing below 700 nm. Transient absorption spectra with excitation at 693 nm exhibit spectral evolution within approximately 2 ps of uphill energy transfer to major spectral forms at 680 nm and downhill energy transfer to red pigments at 705 nm. The 20-30 ps trapping component and P(700) photooxidation spectra derived from data on the 100 ps scale are largely excitation wavelength independent. An additional decay component of red pigments at 710 nm can be induced either by selective excitation of red pigments or by decreasing the temperature to 264 K. This component may represent one of the phases of energy transfer from inhomogeneously broadened red pigments to P(700). The data are discussed based on the available structural model of the photosystem I reaction center and its core antenna.     

Protective dissipation of excess absorbed energy by photosynthetic apparatus of cyanobacteria: role of antenna terminal emitters

Two mechanisms of photoprotective dissipation of the excessively absorbed energy by photosynthetic apparatus of cyanobacteria are described that divert energy from reaction centers. Energy dissipation, monitored as nonphotochemical fluorescence quenching, occurs at different steps of energy transfer within the phycobilisomes or core antenna of photosystem I. Although these mechanisms differ significantly, in both cases, energy dissipates mainly from terminal emitters: allophycocyanin B or core membrane linker protein (L_CM) in phycobilisomes, or the longest-wavelength chlorophylls in photosystem I antenna. It is supposed that carotenoid-induced energy dissipation in phycobilisomes is triggered by light-induced transformation of the nonquenched state of antenna into quenched state due to conformation changes caused by orange carotinoid-binding protein (OCP)–phycobilisome interaction. Fluorescence of the longest-wavelength chlorophylls of photosystem I antenna is strongly quenched by P700 cation radical or by P700 triplet state, dependent on redox state of the acceptor side cofactors of photosystem I.     

Mutations in both sides of the photosystem I reaction center identify the phylloquinone observed by electron paramagnetic resonance spectroscopy

The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.