Hervé Bottin

Time-resolved protein nanocrystallography using an X-ray free-electron laser

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

Ferrodoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803

Abstract The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferrodoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (p I = 3.48), midpoint redox potential ( E m = −0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10 232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (p I = 3.76), close to that of ferrodoxin. The midpoint redox potentials of flavodoxin are E m1 = −0.433 V and E m2 = −0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.

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.

Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus.

Abstract. Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcuselongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential (E′m) of +390 mV in about half of the centers and +275xa0mV in the other half. The high-potential form, whose E′m is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E′m of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E′m when bound to the PSII core (–80xa0mV at pH 6.0) than after its extraction from the complex (–240xa0mV at pH 6.0). The E′m of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58xa0mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.

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.

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.

Laser flash kinetic analysis of Synechocystis PCC 6803 cytochrome c6 and plastocyanin oxidation by Photosystem I

Abstract Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c 6 and plastocyanin to photooxidized P700 in Photosystem I (PS I) particles from the cyanobacterium Synechocystis PCC 6803. Data analysis yields second-order rate constants of 1.3 · 10 7 M −1 s −1 and 1.0 · 10 7 M −1 s −1 for the heme- and copper-proteins, respectively. With the two donor proteins, the observed rate constants ( k obs ) present a linear protein-concentration dependence, thus suggesting an apparent one-step bimolecular collisional mechanism. At neutral pH, the k obs values monotonically increase with increasing NaCl or MgCl 2 concentration, which is ascribed to the involvement of repulsive electrostatic interactions between the donor proteins and PS I. The difference in the effective concentration at which MgCl 2 has its maximum effect as compared with that of NaCl is attributed to the specific role played by Mg 2+ ions, which could act as electrostatic bridges between negatively charged groups. At physiological mild acid pH, cytochrome c 6 is a more efficient electron donor than plastocyanin. The inversion of the NaCl and MgCl 2 effect at pH below 5 — that is, decreasing of k obs with increasing ionic strength — is interpreted as arising from the involvement of attractive ionic interactions at pH lower than the isoelectric point of the donor proteins. Some evolutive aspects on the mechanism of electron donation to PS I are discussed.

Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

The genes encoding (2Fe–2S) plant‐like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedI gene (ssl0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light‐induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150, negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150, sll1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange showed that the C18–C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the FedI carboxy terminus is important for effective PSI/FedI interactions. The plasmid‐shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer.

Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism.