Crystallization and electron paramagnetic resonance characterization of the complex of photosystem I with its natural electron acceptor ferredoxin

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes.     

Pterin-protein complex and ferredoxin as possible components of a molecular complex in photosystem I]

Properties are studied of pterin-protein complex (PPC), possible precursor of ferredoxin in electrone transport chain PS-I. PPC is shown to form chelate complex with Fe ions which are capable to activate the process of light reduction of NADP+ by chloroplasts in the absence of ferrodoxin. A possibility of dark reduction of NADP+ with the participation of light pre-activated PPC is found. Two-step scheme of electrone transport from P700 to ferredoxin is supposed. The data obtained indicate that one of sites of electrone transport into interior part of tilakoid is located on the reduction side of PS-I. An additional possibility of NADP+ reduction with the participation of PPC-Fe in vivo is suggested.     

Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.     

The location of the mobile electron carrier ferredoxin in vascular plant photosystem I

In this study, we present the location of the ferredoxin-binding site in photosystem I from spinach. Image analysis of negatively stained two-dimensional crystals indicates that the addition of ferredoxin and chemical cross-linkers do not significantly alter the unit cell parameters (for untreated photosystem I, a = 26.4 nm, b = 27.6 nm, and gamma = 90 degrees, space group p22(1)2(1) and for ferredoxin cross-linked photosystem I, a = 26.2 nm, b = 27.2 nm, and gamma = 90 degrees, space group p22(1)2(1)). Fourier difference analysis reveals that ferredoxin is bound on top of the stromal ridge principally interacting with the extrinsic subunits PsaC and PsaE. This location would be accessible to the stroma, thereby promoting efficient electron transfer away from photosystem I. This observation is significantly different from that of the ferredoxin binding site proposed for cyanobacteria. A model for the binding of ferredoxin in vascular plants is proposed and is discussed relative to observations in cyanobacteria.     

Importance of the region including aspartates 57 and 60 of ferredoxin on the electron transfer complex with photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

Ferredoxin reduction by photosystem I has been studied by flash-absorption spectroscopy. Aspartate residues 20, 57, and 60 of ferredoxin were changed to alanine, cysteine, arginine, or lysine. On the one hand, electron transfer from photosystem I to all mutated ferredoxins still occurs on a microsecond time scale, with half-times 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 spatially 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.     

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.     

Studies on photosystem I. II. Involvement of ferredoxin in cyclic electron flow

The effects of ferredoxin (Fd) and ferredoxin-NADP reductase on the light-induced spectral changes of cytochrome f (cyt f) were investigated with specific reference to their possible involvement in the cyclic electron transfort pathway of photosystem I (PS I). The steady-state level of photooxidation of reduced cytochrome f is decreased by ferredoxin but unaffected by either ferredoxin-NADP reductase alone or ferredoxin plus ferredoxin-NADP reductase when present in equimolar concentrations. These data are taken as evidence for a cyclic electron transport pathway of: PS I → “X” → Fd → (cyt f) → PC → PS I. The reduced ferredoxin could either reduce directly plastocyanin (PC) or via cytochrome f; the data do not allow differentiation between these two possibilities. However, neither ferredoxin-NADP reductase nor cytochrome b564 appear to serve as electron carriers in this pathway.     

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.     

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 role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP⁺. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI–LHCI–LHCII supercomplex. The binding site(s) of the “additional” LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that “additional” LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

The light-harvesting antennae of photosystem I facilitate energy transfer from trimeric light-harvesting complex II to photosystem I in the stroma lamellae membrane.     

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.     

Reversible inhibition and reactivation of electron transfer in photosystem I

Photosynthesis Research - In photosystem I (PSI) complexes at room temperature electron transfer from A1– to FX is an order of magnitude faster on the B-branch compared to the A-branch. One...     

Electrostatic interactions stabilizing ferredoxin electron transfer complexes. Disruption by "conservative" mutations.

Mitochondrial ferredoxins mediate electron transfer from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes. Previous studies on human ferredoxin, in which acidic residues were replaced with neutral amino acids, established that Asp-76 and Asp-79 are are important for binding to both reductase and P450 (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Here we report that replacement of Asp----Glu at position 76 or 79, whereas maintaining negative charge at these positions also results in dramatic decreases in binding affinity for both electron transfer partners (5-100-fold, delta(delta G) approximately 1.0-2.8 kcal/mol). These results imply that the active electron transfer complexes in these systems are dominated by a stable form which requires specific pairwise electrostatic interactions of fixed geometry for recognition and binding. This mechanism contrasts with that proposed for other electron transfer systems (as exemplified by cytochrome c) in which electrostatic interactions are believed to function primarily in precollisional orientation leading to "encounter complexes" having multiple geometries of similar free energy.     

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.     

Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I.

Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.     

Water-assisted proton transfer in ferredoxin I

The role of water molecules in assisting proton transfer (PT) is investigated for the proton-pumping protein ferredoxin I (FdI) from Azotobacter vinelandii. It was shown previously that individual water molecules can stabilize between Asp(15) and the buried [3Fe-4S](0) cluster and thus can potentially act as a proton relay in transferring H(+) from the protein to the μ(2) sulfur atom. Here, we generalize molecular mechanics with proton transfer to studying proton transfer reactions in the condensed phase. Both umbrella sampling simulations and electronic structure calculations suggest that the PT Asp(15)-COOH + H(2)O + [3Fe-4S](0) → Asp(15)-COO(-) + H(2)O + [3Fe-4S](0) H(+) is concerted, and no stable intermediate hydronium ion (H(3)O(+)) is expected. The free energy difference of 11.7 kcal/mol for the forward reaction is in good agreement with the experimental value (13.3 kcal/mol). For the reverse reaction (Asp(15)-COO(-) + H(2)O + [3Fe-4S](0)H(+) → Asp(15)-COOH + H(2)O + [3Fe-4S](0)), a larger barrier than for the forward reaction is correctly predicted, but it is quantitatively overestimated (23.1 kcal/mol from simulations versus 14.1 from experiment). Possible reasons for this discrepancy are discussed. Compared with the water-assisted process (ΔE ≈ 10 kcal/mol), water-unassisted proton transfer yields a considerably higher barrier of ΔE ≈ 35 kcal/mol.     

Redox potential of quinones in both electron transfer branches of photosystem I

The redox potentials of the two electron transfer (ET) active quinones in the central part of photosystem I (PSI) were determined by evaluating the electrostatic energies from the solution of the Poisson-Boltzmann equation based on the crystal structure. The calculated redox potentials are -531 mV for A1A and -686 mV for A1B. From these results we conclude the following. (i) Both branches are active with a much faster ET in the B-branch than in the A-branch. (ii) The measured lifetime of 200-290 ns of reduced quinones agrees with the estimate for the A-branch and corroborates with an uphill ET from this quinone to the iron-sulfur cluster as observed in recent kinetic measurements. (iii) The electron paramagnetic resonance spectroscopic data refer to the A-branch quinone where the corresponding ET is uphill in energy. The negative redox potential of A1 in PSI is primarily because of the influence from the negatively charged FX, in contrast to the positive shift on the quinone redox potential in bacterial reaction center and PSII that is attributed to the positively charged non-heme iron atom. The conserved residue Asp-B575 changes its protonation state after quinone reduction. The difference of 155 mV in the quinone redox potentials of the two branches were attributed to the conformation of the backbone with a large contribution from Ser-A692 and Ser-B672 and to the side chain of Asp-B575, whose protonation state couples differently with the formation of the quinone radicals.