Light-induced spin polarization in type I photosynthetic reaction centres.

The use of light-induced spin polarization to study the structure and function of type I reaction centres is reviewed. The absorption of light by these systems generates a series of sequential radical pairs, which exhibit spin polarization as a result of the correlation of the unpaired electron spins. A description of how the polarization patterns can be used to deduce the relative orientation of the radicals is given and the most important structural results from such studies on photosystem I (PS I) are summarized. Quinone exchange experiments which demonstrate the influence of protein-cofactor interactions on the polarization patterns are discussed. The results show that there are significant differences between the binding sites of the primary quinone acceptors in PS I and purple bacterial reaction centres and suggest that pi-pi interactions probably play a more important role in PS I. Studies using spin-polarized EPR transients and spectra to investigate the electron transfer pathway and kinetics are also reviewed. The results from PS I, green-sulphur bacteria and Heliobacteria are compared and the controversy surrounding the role of a quinone in the electron transfer in the latter two systems is discussed.

A new approach to determining the geometry of weakly coupled radical pairs from their electron spin polarization patterns.

Analytical expressions for the spin polarized EPR lineshapes of weakly coupled radical pairs (RPs) are derived as functions of the angles between the anisotropic g-tensors of the radicals and the vector describing the dipolar coupling. It is shown that with a singlet precursor the EPR signal of the RP can be written as a linear function of the dipolar coupling. Under these conditions, the calculated powder spectrum can be expressed as a linear combination of four powder spectra, which are independent of the geometry of the RP. To reproduce the experimental spectra the optimal set of coefficients can be found by least-squares fitting. The advantage of this approach is that the four powder spectra must only be calculated once. This treatment shows very clearly the restrictions placed on the information obtainable from such spectra. Most importantly, a unique set of angles can only be obtained if the absolute amplitude of the spectrum is known. In general, the calculated spectrum is related to the experimental spectrum by an unknown, arbitrary scaling factor. In this case, sets of angles consistent with the data are obtained. Possible strategies for obtaining unique geometric information are discussed and demonstrated with the experimental data for the state P+*(865)Q-*(A) in Zn-substituted bacterial reaction centres.

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques.

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t

Recruitment of a foreign quinone into the A(1) site of photosystem I. II. Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy.

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site. These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies.

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

Measurement of cofactor distances between P700.+ and A1.- in native and quinone-substituted photosystem I using pulsed electron paramagnetic resonance spectroscopy.

The radical pair P700.+Q.- (P700 = primary electron donor, Q = quinone acceptor) in native photosystem I and in preparations in which the native acceptor (vitamin K1) is replaced by different quinones is investigated by pulsed EPR spectroscopy. In a two-pulse experiment, the light-induced radical pair causes an out-of-phase electron spin echo, showing an envelope modulation. From the modulation frequency, the dipolar coupling, and therefore the distance between the two cofactors, can be derived. The observation of nearly identical distances of about 25.4 A between P700.+ and Q.- in all preparations investigated here leads to the conclusion that the reconstituted quinones are bound to the native A1 binding pocket. Since the orientation of the reconstituted naphthoquinone relative to the axis joining P700.+ and Q*- differs drastically from that of the native vitamin K1, it cannot be bonded to the protein in the same way as the native acceptor. This implies that the function of A1 as an electron acceptor does not depend on the orientation or hydrogen bonding of the quinone.

The structural organization of the PsaC protein in Photosystem I from single crystal EPR and X-ray crystallographic studies.

In Photosystem I (PS I) the terminal electron acceptors, FA and FB, are iron-sulfur (4Fe-4S) centers, which are bound to the stromal subunit PsaC. The orientation of PsaC is determined relative to the whole PS I complex (see Schubert, W.-D. et al. (1995) in From Light to Biosphere (Mathis, P. ed.), Vol. II, pp. 3-10, Kluwer) from which a molecular model for the structure of PsaC within PS I is derived. Two strategies are followed: (i) PS I single crystal EPR data on the orientation of the g tensors of both FA- and FB- relative to each other and relative to the crystal axes (see preceding paper) are used in conjunction with the central structural part of the bacterial 2 [Fe4S4] ferredoxins, the cysteine binding motifs of which are known to be homologous to those of PsaC; (ii) the same core structure is fitted into the intermediate resolution electron density map of PS I. The PsaC orientation obtained both ways agree well. The local twofold symmetry axis inherent to the ferredoxin model leaves a twofold ambiguity in the structural conclusion. Deviations from this C2-symmetry in the amino acid sequence of PsaC are analyzed with respect to observable properties which would resolve the remaining structural ambiguity. Arguments both for and against FA being the distal iron-sulfur center (to FX) are discussed.

Electron transfer from the acceptor A1 to the iron-sulfur centers in photosystem I as studied by transient EPR spectroscopy.

The electron transfer in photosystem I (PS I) from the secondary acceptor A1 to the iron-sulfur centers is studied by X-band transient EPR with a time resolution of approximately 50 ns. Results are presented for a series of different PS I preparations from the cyanobacterium Synechococcus 6301 ranging from whole cells to core particles in which the iron-sulfur centers have been successively removed. In addition, results from PS I preparations from spinach and the cyanobacterium Synechocystis 6803 are presented. In all samples containing iron-sulfur centers, two consecutive spin-polarized EPR spectra are observed. The two signals have previously been assigned to the charge-separated states P700+.A1-. and P700+.(FeS)-, where (FeS) is one of the three iron-sulfur centers, FX, FA, or FB [Bock, C., van der Est, A., Brettel, K., & Stehlik, D. (1989) FEBS Lett. 247, 91-96]. In agreement with this, the second spectrum is not observed in the sample in which the iron-sulfur centers have been removed. For (P700-FX), core particles which do not contain FA and FB, the second spectrum can unambiguously be assigned to the pair P700+.FX-. In all samples containing FX, the transition from the first to the second spectrum occurs with t1/e approximately 280 ns (t1/2 approximately 190 ns) both in the presence and absence of FA and FB, which strongly suggests that this phase reflects electron transfer from A1-. to FX in intact PS I.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient electron paramagnetic resonance of the triplet state of P700 in photosystem I: evidence for triplet delocalization at room temperature.

Spin-polarized EPR spectra of the triplet state of P700, the primary electron donor in photosystem I (PS I), have been measured for the first time at room temperature. The measurements were performed on intact PS I from Synechococcus sp. after prereduction of all iron-sulfur centers and on vitamin K1 depleted PS I from Synechocystis 6803. The two preparations give similar spectra with a polarization pattern which indicates that the triplet state is formed via recombination of a radical pair. The axial and nonaxial zero-field splitting (zfs) parameters are found to be magnitude of D = (284 +/- 15) x 10(-4) cm-1 and magnitude of E = (22 +/- 3) x 10(-4) cm-1, respectively. The E-value is 42% smaller than in monomeric chlorophyll a, while the D-value is nearly the same. Measurements of the Synechocystis 6803 sample at 4.5 K yielded zfs parameters which are identical with those of the chlorophyll monomer, in agreement with previous results. In order to explain this behavior, it is proposed that the triplet excitation is delocalized over the two halves of a chlorophyll dimer at room temperature but appears localized on one half at low temperature. The observed zfs parameters are obtained if (1) the magnetic z-axes of the two chlorophylls are collinear, (2) the magnetic y-axes (and x-axes) of the two chlorophylls make an angle of approximately 55 degrees with each other, and (3) the admixture of charge-transfer states to 3P700 is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

Nanosecond electron transfer kinetics in photosystem I following substitution of quinones for vitamin K1 as studied by time resolved EPR.

Room temperature transient EPR spectra of photosystem I (PS I) particles from Synechocystis 6803 are presented. Native PS I samples and preparations depleted in the A1-acceptor site by solvent extraction and then reconstituted with the quinones (Q) vitamin K1 (VK1), duroquinone (DQ and DQd12) and naphthoquinone (NQ) have been studied. Sequential electron transfer to P700+A1- (FeS) and P700+A1 (FeS)- is recovered only with VK1. With DQ and NQ electron transfer is restored to form the radical pair P700+Q- as specified by a characteristic electron spin polarization (ESP)-pattern, but further electron transfer is either slowed down or blocked. A qualitative analysis of the K-band spectrum suggests that the orientation of reconstituted NQ in PS I is different from the native acceptor A1 = VK1.