Low temperature (4.2 K) absorption and hole-burned spectra are presented for the trimeric (wild-type, WT) photosystem I complex of the cyanobacterium Synechocystis sp. PCC 6803, its monomeric form, and mutants deficient in the PsaF, K, L, and M protein subunits. High-pressure- and Stark-hole-burning data for the WT trimer are presented as well as its temperature-dependent Q y -absorption and -fluorescence spectra. Taken as a whole, the data lead to assignment of a new and lowest energy antenna Q y -state located at 714 nm at low temperatures. It is this state that is responsible for the fluorescence in the low-temperature limit and not the previously identified antenna Q y -state near 708 nm. The data indicate that the 714 nm state is associated with strongly coupled chlorophyll a molecules (perhaps a dimer) and possesses significant charge transfer character. The red chlorophylls absorbing at 708 and 714 nm do not appear to be directly bound to any of the above protein subunits. The results are consistent with a location close to the interfacial regions between PsaL and M and the PsaA/B heterodimeric core. It is likely that the red chlorophylls are bound to PsaA and/or PsaB.

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Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP͞LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts. Signal recognition particle (SRP) and its receptor comprise essential components of a signal peptide-based protein targeting mechanism that is conserved across evolutionary boundaries (1-3). SRPs in the cytosols of eukaryotes and Escherichia coli target proteins cotranslationally to the endoplasmic reticulum and cytoplasmic membrane, respectively. Targeting is initiated as a result of SRP binding to the hydrophobic domain of amino-terminal signal peptides or signal anchors as they emerge from the ribosome. The entire ribosome͞nascent polypeptide chain complex (RNC) then is piloted by SRP to an SRP receptor that functions at the membrane. GTP binding and hydrolysis by SRP and its receptor result in both the release of SRP from its receptor and the release of SRP from the RNC, whereupon the nascent chain enters a translocation pore that directs the translating polypeptide into or across the lipid bilayer.An organellar SRP, which exhibits striking structural and functional differences from cytosolic SRPs, also has been identified in chloroplasts (4, 5). Chloroplast SRP (cpSRP) is a soluble Ϸ200-kDa stromal particle that contains an evolutionary conserved 54-kDa subunit (cpSRP54) as well as a unique 43-kDa polypeptide (cpSRP43) (6). Unlike cytosolic SRPs, an RNA moiety is conspicuously lacking in cpSRP. Biochemical and genetic evidence have demonstrated that cpSRP functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins belonging to the chlorophyll...

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 E m ؊2 s ؊1 ), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 E m ؊2 s ؊1 ). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50 -60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F A and F B at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c 6 to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A 1 site, where it functions as an efficient cofactor in electron transfer from A 0 to the iron-sulfur clusters.

Electron paramagnetic resonance (EPR) and electronnuclear 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 Å) 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.Light-induced charge separation in all well characterized photosynthetic reaction centers (RCs) 1 proceeds via a common multistep electron transfer process to a stabilized, charge-separated radical pair state P ϩ Q Ϫ consisting of an oxidized (bacterio)chlorophyll donor and a reduced quinone acceptor (whether the green sulfur bacterial RC and the heliobacterial RC contain a quinone acceptor is still controversial). Two types of RCs can be distinguished according to the electron acceptors and electron transfer pathways subsequent to the first quinone acceptor. A series of iron-sulfur clusters with electron transfer essentially perpendicular to the membrane characterize Type I RCs (PS I, green sulfur bacteria, and heliobacteria), whereas a second quinone acceptor Q B and electron transfer parallel to the membrane from the first quinone acceptor Q A characterize Type II RCs (PS II and the RC of purple bacteria). The first quinone is therefore the interface either between electron transfer involving organic cofactors and electron transfer involving iron-sulfur clusters (Type I) or between pure electron transfer and coupled electron/proton tr...

Substituted quinones are usually employed as cofactors in electron transport chains, as demonstrated in bacterial reaction centers (1, 2), and in the two photosystems of plants and cyanobacteria (3-5). These quinones comprise a relatively polar ring, which consists of either a benzoquinone (BQ) 1 or a naphthoquinone (NQ) "head group" and a non-polar isoprenoid "tail" of various chain lengths and degrees of saturation. Benzoquinones such as plastoquinone-9 (PQ-9) and ubiquinone-10 function either as fixed or exchangeable electron/proton carriers during photosynthetic and respiratory electron transport. In photosystem II (PS II), PQ-9 functions as a bound one-electron cofactor in the Q A site and as an exchangeable two-electron/ two-proton cofactor in the Q B site. The reduced PQH 2 -9 is displaced from the Q B site, diffuses laterally through the membrane, and becomes oxidized and deprotonated by the cytochrome b 6 f complex. Photosynthetic reaction centers (RCs) of purple bacteria use either ubiquinone-10 (e.g. Rhodobacter sphaeroides) in a similar double role or menaquinone-9 in the Q A site (e.g. Rhodosprillulum viridis). In photosystem I (PS I), phylloquinone (PhQ), a substituted 1,4-NQ with a 20-carbon, largely saturated phytyl tail, functions as a bound one-electron cofactor in the A 1 site. PS I contains two PhQ molecules/P700, but neither of the two quinones functions in a manner equivalent to Q B in the bacterial RC and PS II. Instead, the electron is transferred from the active quinone(s) to soluble ferredoxin via a chain of three bound iron-sulfur centers. Quinones are therefore extremely versatile; they can function as the interface between electron transfer involving organic cofactors and electron transfer involving iron-sulfur clusters (as in PS I), or between pure electron transfer and coupled electron/proton transfer involving a second organic cofactor (as in PS II). Each quinone displays equilibrium binding and redox properties that can be very different for each site of interaction (6), and these properties are conferred largely by the protein environment.To understand the structural determinants that allow quinones to function with a low redox potential in the A 1 site of PS I, we embarked on a project aimed at biological replacement of

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.

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