Örjan Hansson

Current perceptions of Photosystem II.

In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385–398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.

The Origin of the Multiline and g=4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).

Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I

Plastocyanin (Pc) has been modified by site‐directed mutagenesis at two separate electron‐transfer (ET) sites: Leu‐12‐Glu at a hydrophobic patch, and Tyr‐83‐His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu‐12‐Glu) and increased by 35 mV in Pc(Tyr‐83‐His). The latter mutant shows a 2‐fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu‐12‐Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.

Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin

The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the northern hydrophobic patch and Tyr-83 and Asp-42 in the eastern acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.

Kinetic evidence for the function of Z in isolated photosystem II reaction centers

(D1,D2) Photosystem II reaction centers were studied by flash absorption spectroscopy with microsecond resolution. Without addition, absorption transients in the 660–850 nm region are due to a triplet state, probably of P‐680, with a decay half‐time of 700 μs (no oxygen) or 40 μs (under air). With the addition of DBMIB, presumably acting as electron acceptor, new kinetic and spectral features appear, which are attributed to P‐680. A 5 μs phase of decay is present, which is pH‐dependent and is attributed to donation from Z to P‐680+.

Charge recombination between stabilized P-680+ and reduced cytochrome b-559 in quinone-reconstituted PS II reaction center

Abstract Flash-induced absorbance changes in the nanosecond to millisecond time ranges have been measured in the Photosystem II reaction center complex consisting of D1 and D2 subunits and cytochrome b -559, in the presence of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). The results indicate that DBMIB largely quenches the primary radical pair (P-680 + Pheo − ) and the formation of the triplet ( 3 P-680). Long-lived absorption signals in the red and near-infrared (bleaching at 680 nm and broad increase at 740–830 nm) and in the green (peak at 560 nm) can be attributed to the oxidation of P-680 and to the reduction of cytochrome b -559. These data show that addition of DBMIB induces stabilization of P-680 + and a rapid (perhaps submicrosecond) reduction of cytochrome b -559. The signals attributed to P-680 + and to reduced cytochrome decay in parallel ( t 1 2 = 2 ms ), showing that the cytochrome reduces P-680 + . The stabilization occurred also in the presence of plastoquinone-3 and (with DBMIB) at −29°C in a viscous solution containing 60% glycerol at a low concentration of quinone, suggesting that the quinone reconstitutes the function of Q A and thus mediates electron transport from the reduced pheophytin a to the intrinsic cytochrome.

The interaction of ammonia with the photosynthetic oxygen-evolving system

Abstract The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S2 state and believed to be accessible in this state only, was found to bind ammonia also in the S1 state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S1 and S2 states. The apparent dissociation constants for ammonia at the two sites in the S1 and S2 states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of 17O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S2 state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.

Flash photolysis studies of manganese-depleted Photosystem II: evidence for binding of Mn2+ and other transition metal ions

The reduction rate of P680 + , the oxidized form of the primary electron donor of Photosystem II, has been studied with flash absorption spectroscopy at 830 nm. Photosystem II membranes, partially depleted of the intrinsic managanese of the oxygen-evolving complex, were used. The reduction rate of P680 + was measured as a function of the concentration of free Mn 2+ , which was stabilized by metal-ion buffer systems consisting of chelators and metal-chelator complexes. Increasing the Mn 2+ concentration induced an 18 to 35 μs decay component in the P680 + reduction kinetics and diminished the amplitude of the 4 to 8 μs decay component. A dissociation constant of approx. 50 μM was obtained for the observed Mn 2+ binding site. Other transition metal ions affected the reduction kinetics of P680 + at lower concentrations. Thus, photooxidation of Mn 2+ is not required for the detection of its binding at this site. To account for the kinetic effect, it is proposed that the bound metal ion interacts electrostatically with the tyrosine residue Y Z , the intrinsic electron donor to P680 + .

The involvement of the two acidic patches of spinach plastocyanin in the reaction with photosystem I

Six different spinach plastocyanin mutants have been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the two acidic patches in the interaction with photosystem I. The mutants were: Asp42Lys, Glu43Asn, Glu43Lys, Glu43Gln/Asp44Asn, Glu59Lys/Glu60Gln and Glu43Asn/Glu59Lys/Glu60Gln and they have been characterised by optical absorption and EPR spectroscopy, redox titrations and isoelectric focusing. The electron transfer to photosystem I was investigated by flash-induced time-resolved absorption measurements at 830 nm. The kinetics were interpreted with a model that incorporates a rate-limiting conformational change from inactive to active forms of the plastocyanin-photosystem I complex. All mutations resulted in a displacement of the equilibrium towards the inactive conformation. The strongest impairment of the electron transfer was found for mutations in the larger acidic patch, in particular upon modification of residues 43 or 44. However, mutations of residues 59 and 60 in the smaller acidic patch also resulted in a lower reactivity.

Spectroscopic and kinetic characterization of the spinach plastocyanin mutant Tyr83-His: a histidine residue with a high pK value

Tyrosine-83 in spinach plastocyanin (Pc) has been modified by site-directed mutagenesis to a histidine. An NMR titration yields a pK value of 8.44 for this residue. The high value is probably due to the acidic residues close to this site. The reduction potential is increased by 35 mV at pH 7.5, but only slightly, if at all, at pH 8.9. EPR and optical absorption bands associated with the copper site are not affected by the mutation, either at pH 7.5 or at pH 8.9. The electron transfer (ET) to Photosystem I (PS I), as studied by a flash-photolysis technique, is pH dependent for the mutant, being slower than the wild type at pH 7.5 but more similar to it at pH 8.9. The data have been interpreted with a model that includes a rate-limiting conformational change in the Pc-PS I complex which precedes the intracomplex ET (Bottin, H. and Mathis, P. (1985) Biochemistry 24, 6453-6460). The slower kinetics at the lower pH for the mutant is attributed to a dual effect of the protonation of the His-83 residue: (i) A destabilization of the close bound conformation, i.e., the one competent in electron transfer, and (ii) a smaller intracomplex ET rate constant, partly due to a smaller driving force for ET. From this it is concluded that the Tyr-83 residue is not a part of the ET pathway to PS I.