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

Photosystem I has two branches of cofactors down which lightdriven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (F X) is described by two kinetic components with decay times of Ϸ20 and Ϸ200 ns, which have been proposed to represent ET from PhQ B and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to F X. The mutation near ec3 A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors.Chlamydomonas ͉ directionality ͉ photosynthetic reaction center ͉ pump-probe spectroscopy ͉ transient EPR P hotosynthetic reaction centers (RCs) are the membrane proteins responsible for the capture and storage of light energy in photosynthetic organisms. As with all of the RCs, Photosystem I (PS1) has a C2 symmetrical structure with two virtually identical branches of redox cofactors extending across the membrane (see Fig. 1). The x-ray crystal structure of PS1 from Thermosynechococcus elongatus (1) has allowed identification of amino acid residues interacting with the electron transfer (ET) cofactors, permitting the use of site-directed mutagenesis to investigate to what extent ET occurs in the two branches. Many of these studies (2-10) point toward the possibility that ET takes place in both branches of cofactors (A-branch and Bbranch; see Fig. 1). The data consistently show that the major fraction occurs in the A-branch and that the slow phase of ET from phylloquinone (PhQ) to F x is associated with this branch. The involvement of the B-branch is less clear, but there is mounting evidence to support the assignment of the fast component of PhQ to F x ET to this branch. If ET does indeed occur in both branches in PS1, this behavior would be remarkably different from the type II RCs. In purple bacterial RCs, for example, it is well known that initial ET is biased almost exclusively toward the A-branch, probably because the B-branch quinone is a mobile electron and proton carrier in this type of RC. The strong biasing of the ET in the type II RCs makes it difficult to investigate the fac...

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“…These effects are well documented experimentally for the chlorobial RC (27) and the PS1 ec3 axial ligand mutants (ref. 9 and reviewed in ref.…”

Section: Discussionsupporting

confidence: 56%

Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

Est

Lucas

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

101

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“…Transient EPR Spectroscopy at X-band and Q-Band-Low temperature, X-band (9-GHz) transient EPR experiments were performed with a laboratory-built spectrometer using a Bruker ER046 XK-T microwave bridge equipped with an ER-4118X-MD-5W1 dielectric ring resonator and using an Oxford CF935 helium gas flow cryostat (25). The loaded Q value for this dielectric ring resonator was about 3000, equivalent to a rise time of r ϭ Q/(2* mw ) Ϸ 50 ns.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

Sakuragi

Zybailov

Shen

et al. 2005

Journal of Biological Chemistry

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A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of F X , F B , and F A was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700؉ measured in the near-IR and the decay of A 1 ؊ measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 s. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700؉ and PQ-9 ؊ . The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A 1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3 P700 product of P700 ؉ A 0 ؊ charge recombination via the T 0 spin level in those PS I complexes that do not contain a functional quinone. In those A 1 sites that are occupied, the P700 ؉ Q ؊ polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A 1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A 1 site in the menB rubA mutant due to the absence of F X and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A 1 site by washing. However, all A 1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A 1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I.Photosystem I is a multisubunit, pigment-protein complex that is found in the membranes of plants, algae, and cyanobacteria and that mediates the light-induced transfer of electrons from plastocyanin/cytochrome c 6 to ferredoxin/flavodoxin. According to current understanding, light-induced charge separation results in the oxidation of the primary electron donor P700 (E m Ј ϩ430 mV), a chlorophyll a/aЈ heterodimer located on the luminal (inner) side of the membrane, and the reduction of the primary electron acceptor A 0 (E m Ј approximately Ϫ1000 mV), a chlorophyll a monomer located in the interior of the membrane. The electron is passed to A 1 (E m Ј approximately Ϫ800 mV), an alkyl-substituted menadione (2-methyl-1,4-naphthalenedione); to F X (E m Ј Ϫ705 mV), an interpolypeptide (4Fe-4S) cluster; and finally to F A (E m Ј Ϫ520 mV) and F B (E m Ј Ϫ580 mV), which are (4Fe-4S) clusters bound to the extrinsic subunit PsaC located on the stromal (cytoplasmic) side of the membrane. In most organisms, including Synechocystis sp. PCC 6803, the quinone in the A 1 site is phylloquinone (2-methyl-3-phytyl-1,4-naphtho...

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“…Variable Temperature X-band, Q-band, and W-band Transient EPR-The low temperature X-band (9 GHz) transient EPR experiments were carried out on a laboratory built spectrometer using a Bruker ER046 XK-T microwave bridge equipped with an ER-4118X-MD-5W1 dielectric ring resonator and an Oxford CF935 helium gasflow cryostat (18). The loaded Q-value for this dielectric ring resonator was about Q ϭ 3000, equivalent to a rise time of r ϭ Q/(2 ϫ mw ) Ϸ 50 ns.…”

Section: Methodsmentioning

confidence: 99%

Electron Transfer in Cyanobacterial Photosystem I

Chitnis

Valieva

et al. 2003

Journal of Biological Chemistry

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The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp A 1؊ radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697F PsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692C PsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P 700 ؉ A 1 ؊ and the CW EPR spectra of photoaccumulated A 1 ؊ . We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P 700 and A 1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR. Photosynthetic reaction centers (RCs)1 are classified into two general types depending on the identity and function of the terminal electron acceptors. Those RCs that incorporate ironsulfur clusters are classified as "Type I," and those that incorporate a mobile (secondary) quinone are classified as "Type II." Type I RCs include Photosystem I (PS I) of cyanobacteria and plants and those in heliobacteria and green sulfur bacteria. Type II RCs include Photosystem II of cyanobacteria and plants and those in green non-sulfur bacteria and purple bacteria. Despite the difference in the identity of the terminal electron acceptors, Type I and Type II RCs share a common motif in terms of polypeptide arrangement and cofactor composition (1).The primary cofactors are bound to proteins that are present as dimers in the membrane. This results in a set of electron transfer cofactors that are arranged (pseudo)symmetrically (2). In PS I, these cofactors include a special pair of chlorophyll a/aЈ molecules as the primary donor, two bridging chlorophyll a molecules, and two chlorophyll a molecules, at least one of which functions as the primary acceptor (3). In the purple bacterial reaction center, the cofactors include a special pair of bacteriochlorophyll a molecules as the primary donor, two bridging bacteriochlorophyll a molecules, and two pheophytin molecules, one of which functions as the primary acceptor (4). PS I and the purple bacterial reaction center contain two quinones; in the latter, one quinone is rather immobile (Q A ) and the other is mobile (Q B ), whereas in PS I both quinones (Q K -A and Q K -B) are, to the best of our knowledge, immobile in their normal function.In Type II reaction centers, a single turnover results in the reduction of Q B , to a semiquinone and a second turnover results in the further reduction (and protonation) of Q B to a hydroquinone. The stability of Q B Ϫ requires that t...

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

Cited by 56 publications

References 49 publications

Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

Electron Transfer in Cyanobacterial Photosystem I

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