Pierre Sétif

Flash‐induced absorption changes in photosystem I at low temperature: evidence that the electron acceptor A1 is vitamin K1

Low temperature flash absorption spectroscopy has been applied to elucidate the chemical nature of the secondary electron acceptor A1 of photosystem I (PS‐I). The flash‐induced absorption changes measured in digitonin‐fractionated spinach PS‐I particles at 10 K between 240 and 525 nm are shown to comprise a major decay phase with t ~ 150 μs which has been attributed to the recombination reaction P‐700+·A1 → P‐700·A1 [(1984) Biochim. Biophys. Acta 767, 404‐414]. We present the absorption difference spectrum of this reaction and demonstrate that it contains contributions in the ultraviolet due to A1, which are characteristic of vitamin K1 (phylloquinone).

Ferredoxin and flavodoxin reduction by photosystem I.

Ferredoxin and flavodoxin are soluble proteins which are reduced by the terminal electron acceptors of photosystem I. The kinetics of ferredoxin (flavodoxin) photoreduction are discussed in detail, together with the last steps of intramolecular photosystem I electron transfer which precede ferredoxin (flavodoxin) reduction. The present knowledge concerning the photosystem I docking site for ferredoxin and flavodoxin is described in the second part of the review.

The ferredoxin docking site of photosystem I.

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 A resolution [Nature 411 (2001) 909]. 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.

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Abstract Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t 1 2 ≈ 120 μ s . When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component ( t 1 2 = 50–400 ms ) reflecting the recombination between P-700+ and center X− occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700+ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A−1 as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.

The PsaC subunit of photosystem I provides an essential lysine residue for fast electron transfer to ferredoxin

PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. 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 K35 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 K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash‐absorption spectroscopy, whereas the K35R 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 K35 by T, D, E or R abolishes Fd cross‐linking to PsaC, and cross‐linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross‐linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd.

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross‐linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10–14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20–25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe‐ 2S] center of ferredoxin and the terminal [4Fe‐4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.

Tentative identification of the apoproteins of iron-sulfur centers of Photosystem I

A newly purified Photosystem (PS) I particle is described, with still active iron‐sulfur acceptors: A, B and X. Apart from the apoprotein of P700, 3 other main polypeptides of this particle are located at 20, 17 and 10 kDa, and two minor ones are detectable at 16.5 and 8 kDa. Both in vivo 35S labeling and carboxymethylation with iodo[14C]acetate show that most of the cysteine residues are located in the 8‐kDa band. The amino acid composition of this band reveals important common features with small iron‐sulfur proteins of the ferredoxin type.

Flash-induced absorption changes in Photosystem I, Radical pair or triplet state formation?

Abstract Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700+ ... A−1 (A1 being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700+. A saturation curve (ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700+. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700+ and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.

Light-induced charge separation in photosystem I at low temperature is not influenced by vitamin K-1

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.

Absorption studies of Photosystem I photochemistry in the absence of vitamin K-1

Abstract Flash-induced absorption changes were studied on different timescales (nanosecond to millisecond) and at different temperatures (10 to 278 K) in highly enriched spinach PS I particles lacking vitamin K-1 and in which the electron transfer from the primary acceptor to the secondary acceptors was blocked. At all temperatures, the initial absorption change at 820 nm was followed by a fast decay ( t 1 2 ≈ 47 ns at 278 K and ≈ 82 ns at 10 K) which is attributed to the decay of the primary radical pair (P-700+-A−0). A slower phase of absorption decay is attributed to the P-700 triplet state, which was formed as a result of the biradical recombination, with a yield of about 30% at 278 K and about 75% at 10 K. Under air, the 3P-700 state decayed with a t 1 2 of about 50 μs at 278 K, whereas in the absence of oxygen it decayed with t 1 2 ≈ 560 μs . At 278 K, this yield was shown to depend on the presence of a magnetic field, with a maximum around 60 G. The 3P-700 decay halftime was nearly independent of temperature in the absence of oxygen ( t 1 2 ≈ 1 ms at 10 K ). The implications for the mechanisms involved in this decay are discussed. Addition of vitamin K-1 to these particles resulted in a decrease in the amplitude of the fast submicrosecond decay and a concomitant increase in the amplitude of a slow phase, indicating an efficient transfer from A−0 to vitamin K-1. However, most functional properties of the acceptor A1 were not reconstituted under these conditions.