Discovery of Native Metal Ion Sites Located on the Ferredoxin Docking Side of Photosystem I (original) (raw)
Related papers
The ferredoxin docking site of photosystem I
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 Å resolution [Nature 411 ]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity. D
Biochemical and Biophysical Research Communications, 2000
Ferredoxin reduction by photosystem I has been studied by flash-absorption spectroscopy. Aspartate residues 20, 57, and 60 of ferredoxin were changed to alanine, cystein, arginine, or lysine. On the one hand, electron transfer from photosystem I to all mutated ferredoxins still occurs on a microsecond time scale, with halftimes of ferredoxin reduction mostly conserved compared to wild-type ferredoxin. On the other hand, the total amplitude of the fast first-order reduction varies largely when residues 57 or 60 are modified, in apparent relation to the charge modification (neutralized or inverted). Substituting these two residues for lysine or arginine induce strong effects on ferredoxin binding (up to sixfold increase in K D ), whereas the same substitution on aspartate 20, a spacially related residue, results in moderate effects (maximum twofold increase in K D ). In addition, double mutations to arginine or lysine were performed on both aspartates 57 and 60. The mutated proteins have a 15-to 20-fold increased K D and show strong modifications in the amplitudes of the fast reduction kinetics. These results indicate that the acidic area of ferredoxin including aspartates 57 and 60, located opposite to the C-terminus, is crucial for high affinity interactions with photosystem I.
Biophysical Journal, 2005
Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q B . On the basis of x-ray diffraction at 2.5 Å resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd 21 or Zn 21 . Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn 21 ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn 21 binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by ;2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.
Biochemistry, 2005
Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganesebinding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highestaffinity site had a dissociation constant of approximately 1 µM. For the highest-affinity mutant, a firstorder rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 µM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex. Pigment-protein complexes convert light energy into chemical energy in photosynthetic organisms. In purple anoxygenic bacteria, reaction centers embedded in the membrane perform light-driven charge separation (1). The primary electron donor of the reaction center from Rhodobacter sphaeroides is a bacteriochlorophyll a dimer (P) 1 which upon excitation becomes oxidized through electron † This work was supported by Grant MCB 0131764 from the National Science Foundation. ‡ The coordinates and structure factors for the structures reported here are available from the Protein Data Bank (entries 1Z9K and 1Z9J for the wild type and mutant, respectively).
The Journal of biological chemistry, 1991
Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive t...
J Mol Biol, 2001
Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloro¯exus aurantiacus, crystallizes in space group P6 4 22 (a b 115.7 A Ê , c 54.6 A Ê). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was re®ned at 1.55 A Ê resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H 2 O molecules, one Cl À and two SO 4 2À. The ®nal residual and estimated standard uncertainties are R 0.198, ESU 0.076 A Ê for atomic coordinates and ESU 0.05 A Ê for CuÐligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits signi®cant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.
The EMBO Journal, 1998
PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors F A and F B. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K 35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K 35 T, K 35 D and K 35 E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K 35 R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K 35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K 35 T, K 35 D and K 35 E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K 35 is an interaction site between PsaC and its redox partner Fd.
Journal of Molecular Biology, 2001
Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloro¯exus aurantiacus, crystallizes in space group P6 4 22 (a b 115.7 A Ê , c 54.6 A Ê ). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was re®ned at 1.55 A Ê resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H 2 O molecules, one Cl À and two SO 4 2À . The ®nal residual and estimated standard uncertainties are R 0.198, ESU 0.076 A Ê for atomic coordinates and ESU 0.05 A Ê for CuÐligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits signi®cant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.