Early Electron Acceptors Research Articles

Picosecond transient absorption spectroscopy in the blue spectral region of photosystem I.

Picosecond transient absorption difference spectroscopy in the blue wavelength region (380-500 nm) was used to study the early electron acceptors in photosystem I. Samples were photosystem I core particles with about 100 chlorophylls per reaction center isolated from the cyanobacterium Synechocystis sp. PCC 6803. After excitation at 590 nm at room temperature, decay-associated spectra (DAS) were determined from global analysis in the blue region, yielding two transient components and one nondecaying component. A 3 ps decay phase is interpreted as primarily due to antenna excited-state redistribution. A 28 ps decay phase is interpreted as due to overall excited-state decay by electron transfer. The nondecaying component is ascribed to the difference spectrum of P(700) and the quinone or A(1) electron acceptor (P(700)(+)A(1)(-) - P(700)A(1)). Decay curves on the millisecond time scale at different wavelengths were measured with an autoxidizable artificial electron acceptor, benzyl viologen, and the (P(700)(+) - P(700)) difference spectrum was constructed. The (A(1)(-) - A(1)) difference spectrum was obtained by taking the difference between the above two difference spectra. A parallel picosecond experiment under strongly reducing conditions was also done as a control experiment. These conditions stabilize the electron on an earlier acceptor, A(0). The nondecaying component of the DAS at low potential was assigned to (P(700)(+)A(0)(-) - P(700)A(0)) since the electron-transfer pathway from A(0) to A(1) was blocked. The [(P(700)(+)A(0)(-) - P(700)A(0)) - (P(700)(+) - P(700))] subtraction gives a spectrum, interpreted as the (A(0)(-) - A(0)) difference spectrum of a chlorophyll a molecule, consistent with previous studies. The (A(1)(-) - A(1)) spectrum resolved on the picosecond time scale shows significant differences with similar spectra measured on longer time scales. These differences may be due to electrochromic effects and spectral evolution.

Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A0, A1 and Fe-SX. Fe-SX is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-SX is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-SA/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.

Photosystem I core

Recent work on the structure of photosystem I (PS I) is reviewed in historical context. Composition and methods of preparation of the reaction center, core and antennae are described. Emphasis is on the polypeptide composition of the various parts of the photosystem, especially the core, which also includes the reaction center. Recent work on the assignment of iron-sulfur centers to particular polypeptides is described as well as work on other early electron acceptors. The interactions of PS I with ferredoxin (which forms the link to pyridine nucleotide reduction) and with plastocyanin (which forms the link with the cytochrome b 6/f complex) are presented. Reports on the sequences of chloroplast- and nuclear-coded polypeptide components of PS I are summarized along with interpretations of the structure of the photosystem derived from such results. Work on attempts to determine structure at the atomic level by X-ray crystallography is described. The relationship of green plant and cyanobacterial PS I to various types of bacterial photosynthetic pigment-protein complexes is discussed. The protein-dimer nature of PS I reaction center is reviewed and evaluated. Data suggesting that the isolated green plant PS I reaction center may not be a protein-dimer are presented.

The photosystem of the green sulfur bacterium Chlorobium limicola contains two early electron acceptors similar to photosystem I

An asymmetric EPR-line with a g factor of 2.0045 was detected after phototrapping at 196 K in membranes and in isolated photoactive P840 reaction centers of Chlorobium limicola f. thiosulfatophilum. The spectra resemble those of the electron acceptor A − 1 of photosystem I in chloroplasts and cyanobacteria. At 229 K a symmetric signal at g=2.0033, comparable to the one of the early electron acceptor A − 0 is additionally phototrapped. In contrast to membranes, the reaction center preparation does not contain appreciable amounts of photoreducible FeS-centers.

Electron transport in green photosynthetic bacteria

Green bacteria make up two of the four families of anoxygenic photosynthetic prokaryotes. The two families have similar pigment compositions and membrane fine structure, and both contain a specialized antenna structure known as a chlorosome. The primary photochemistry and electron transport pathways of the two groups are, however, quite distinct. The anaerobic green bacteria (Chlorobiaceae) contain low-potential iron-sulfur proteins as early electron acceptors and can directly reduce NAD(+) in a manner reminiscent of Photosystem I of oxygenic organisms. The facultatively aerobic green bacteria (Chloroflexaceae) contain quinone-type acceptors and have an overall pattern of electron transport very similar to that found in purple bacteria. Many aspects of energy storage in green bacteria, especially photophosphorylation and the role of cytochrome b/c complexes in electron transport, remain poorly understood.

Picosecond measurements of the primary photochemical events in reaction centers isolated from the facultative green photosynthetic bacterium Chloroflexus aurantiacus: Comparison with the purple bacterium Rhodopseudomonas sphaeroides

The primary charge separation process in reaction centers isolated from the green photosynthetic bacterium Chloroflexus aurantiacus has been investigated with picosecond transient absorption techniques. Transient spectra and kinetics are compared with those obtained under identical conditions on reaction centers from the purple bacterium Rhodopseudomonas sphaeroides. An early electron acceptor complex in both organisms appears to involve interacting bacteriopheophytin- a and bacteriochlorophyll- a molecules. However, there are significant spectral and kinetic differences in the two reaction center preparations.

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus.

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.

The effect of ambient redox potential on the transient electron spin echo signals observed in chloroplasts and photosynthetic algae

Time-resolved electron spin echo (ESE) studies were carried out at room temperature on chloroplast preparations and whole cells of photosynthetic algae. The signals observed exhibit the unexpected special ESE signal which we have proposed to be the result of transient interactions between P +-700 and an early electron acceptor of Photosystem I (Thurnauer, M.C. and Norris, J.R. (1980) Chem. Phys. Lett. 76, 557–561). The intensity of the special ESE signal decreases with the chemical reduction of the Center A-Center B complex. The results suggest that in the untreated photosynthetic systems we are initially observing P +-700 as it interacts with the reduced acceptor which precedes the Center A-Center B complex. Then the decay of the special ESE signal (approx. 170 ns) gives the lifetime of this reduced acceptor as it participates in forward electron transport.

Triplet states of monomeric bacteriochlorophyll in vitro and of bacteriochlorophyll dimers in antenna and reaction center complexes

Triplet states of monomeric bacteriochlorophyll in solution, and of dimeric bacteriochlorophyll in photosynthetic reaction centers and antenna complexes isolated from Rhodopseudomonas sphaeroides and Rps. viridis were generated by excitation with short flashes. The triplet states were characterized by measurements of optical absorption spectra, decay kinetics as functions of temperature, quenching by O 2, and delayed fluorescence. The triplet state of the antenna dimer can be described as one molecule in the ground (singlet) state and one in the excited (triplet) state. Formation of the triplet disrupts exciton interactions between the two molecules, and the complex acquires a new absorption band due to the molecule in the ground state. In reaction centers, the formation of the triplet causes bleaching of the main absorption band of P, the reactive bacteriochlorophyll complex (870 nm in Rps. sphaeroides; 960 nm in Rps. viridis). Other absorption bands that have been considered to be exciton bands of P (810 nm in Rps. sphaeroides; 850 nm in Rps. viridis) appear to undergo shifts, but not to bleach. The latter bands probably are due to neighboring bacteriochlorophylls, not to P. The decay kinetics of the triplet state in reaction centers are strongly influenced by interactions with the anionic radical of the quinone that serves as an early electron acceptor. A new transient (20 ns) state of the reaction center was found when reaction centers were excited with supersaturating flashes.

Nanosecond fluorescence and absorbance changes in photosystem II at low redox potential: Pheophytin as an intermediary electron acceptor

The electron-acceptor system in photosystem II (PS II) reaction centers of green plants is similar to that in bacterial reaction centers. In both kinds of reaction centers, a quinone (Q) acts as an early electron acceptor (ubiquinone or menaquinone in bacterial [l] and plastoquinone in PS II [2,3]). If the quinone is in the reduced form, continuous illumination causes the accumulation of a bacteriopheophytin (BPh) radical anion (BPh’-) in bacterial reaction centers [4-61 and of a pheophytin (Ph) radical anion (Ph.-) in PS II [7-l 31. The radical anions of BPh [5,14,15] and of Ph [12,13] participate inan exchange interaction with the radical anion of the quinone, coupled to a non-heme Fe atom, indicating that the distance between (B)Ph and Q is very short. The reduction of the quinone increases the formation of pigment triplet states in both bacteria [4,16] and PS II [29]. This is inhibited upon photoaccumulation of BPH’or Ph.-. Reduction of Q also is accompanied by the appearance of ns delayed fluorescence, which decreases during photoaccumulation of BPh’[4] or Ph.[9]. The delayed fluorescence has a characteristic magnetic field dependence in bacterial reaction centers [ 17-191 and in PS II [ 181 and an activation energy of 0.04-0.12 eV [4,9]. When the quinone in bacterial reaction centers is reduced, flash excitation generates a state ‘PF’, which has a lifetime of -10 ns [ 16,201. The transient state

Spectral and kinetic evidence for two early electron acceptors in photosystem I

Triton-fractionated photosystem-I particles poised at -625 mV, where the two bound iron-sulfur proteins are reduced, have been studied by optical and electron paramagnetic resonance spectroscopies from 293 to 5 K. At 5-9 K, these particles exhibit two decay components with lifetimes of 1.3 and 130 msec in the laser pulse-induced absorption and electron paramagnetic resonance signal changes. Spectral properties of the 130-msec decay component reflect the charge separation between P-700 and some iron-sulfur center having a broad optical absorbance in the 400- to 550-nm region and a previously reported electron paramagnetic resonance signal with g = 1.78, 1.88, and 2.08. Spectral properties of the 1-msec decay component indicate photoinduced charge separation between P-700 and a chlorophyll a dimer having absorption bands at 420, 450, and 700 nm. It is assumed that these two acceptors participate in the electron transfer from P-700(*) to the bound iron-sulfur proteins.

Development of electron spin polarization in photosynthetic electron transfer by the radical pair mechanism

We have extended the radical pair theory to treat systems of membrane-bound radicals with g tensor anisotropy. Analysis of the polarized electron paramagnetic resonance (EPR) signals of P700+, originating from photosystem I of higher plants, in terms of the radical pair mechanism provides information about the sequence of early electron acceptors. To account for the orientation dependence of the line shape and integrated area of this polarized signal, we propose the electron transfer sequence to be P700 leads to A1 leads to X leads to Fd(A, B), where A1 is a small organic molecule (possibly chlorophyll), X is the acceptor species observed recently in low-temperature EPR studies, and Fd(A, B) are the ferredoxin iron-sulfur centers A and B. Our calculations provide information about the life-times of A1-, and X-, and their exchange interactions with P700+. We also find supporting evidence for the orientation of X- in the thylakoid membrane reported recently by G. C. Dismukes and K. Sauer (Biochim. Biophys. Acta. 504:431-445.).

The orientation of membrane bound radicals. An EPR investigation of magnetically ordered spinach chloroplasts

The orientation of membrane-bound radicals in spinach chloroplasts is examined by electron paramagnetic resonance (EPR) spectroscopy of chloroplasts oriented by magnetic fields. Several of the membrane-bound radicals which possess g-tensor anisotropy display EPR signals with a marked dependence on the orientation of the membranes relative to the applied EPR field. The fraction of oxidized and reduced plastocyanin, P-700, iron-sulfur proteins A and B, and the X center, an early acceptor of Photosystem I, can be controlled by the light intensity during steady-state illumination and can be trapped by cooling. The X center can be photoreduced and trapped in the absence of strong reductants and high pH, conditions previously found necessary for its detection. These results confirm its role as an early electron acceptor in P-700 photo-oxidation. X is oriented with its smallest principal g-tensor axis ( g x) predominantly parallel to the normal to the thylakoid membrane, the same orientation as was found for an early electron acceptor based on time-resolved electron spin polarization studies. We propose that the X center is the first example of a high potential iron-sulfur protein which functions in electron transfer in its ‘;superreduced’; state. We present evidence which suggests that iron-sulfur proteins A and B are 4Fe-4S clusters in an 8Fe-8S protein. Center B is oriented with g y predominantly normal to the membrane plane. The spectra of center A and plastocyanin do not show significant changes with sample orientation. In the case of plastocyanin, this may indicate a lack of molecular orientation. The absence of an orientation effect for reduced center A is reconcilable with a 4Fe-4S geometry, provided that the electron obtained upon reduction can be shared between any pair of Fe atoms in the center. Orientation of the ‘;Rieske’; iron-sulfur protein is also observed. It has axial symmetry with g ∥ close to the plane of the membrane. A model is proposed for the organization of these proteins in the thylakoid membrane. A new EPR signal was observed in oriented chloroplasts. This broad unresolved resonance displays a g value of 3.2 when the membrane normal is parallel to the field. It shifts to g = 1.9 when the membrane normal is perpendicular to the field. The signal is sensitive to illumination and to washing of the thylakoid membranes of broken chloroplasts. We suggest that there is a relation between this signal and the water-oxidizing enzyme system.