Pulsed EPR Study of Spin-Coupled Radical Pairs in Photosynthetic Reaction Centers:  Measurement of the Distance Between   and   in Photosystem I and between   and   in Bacterial Reaction Centers

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The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane ␣-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A 1 ؊ ) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp 693 to Phe resulted in an inability to photo-accumulate A 1 ؊ , whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp 673 ) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P 700 ؉ A 1 ؊ radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A 1 ؊ phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp 693 . However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.

Section: Methodsmentioning

confidence: 99%

Mutations in Both Sides of the Photosystem I Reaction Center Identify the Phylloquinone Observed by Electron Paramagnetic Resonance Spectroscopy

Boudreaux¹,

MacMillan²,

Teutloff³

et al. 2001

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“…The echo mTmodulation was recorded at 512 points with 8-ns increments in , and 64 traces were averaged to increase the signal/noise ratio (see Ref. 13 for details).…”

Section: Insertion Of the [4fe-4s] Cluster Into The F X Site In Ps Imentioning

confidence: 99%

Assembly of Photosystem I

Shen

Antonkine

Est

et al. 2002

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The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, F X , F A , and F B , are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubA mutant. The flashinduced decay kinetics of both P700 ؉ in the visible and A 1 ؊ in the near-UV show that charge recombination occurs between P700؉ and A 1 ؊ in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A 1 . In agreement, the spin-polarized X-band EPR spectrum of P700 ؉ A 1 ؊ at low temperature shows that an electron cycle between A 1 ؊ and P700 ؉ occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [F A /F B ]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700 ؉ to A 1 ؊ center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of F X , F B , and F A , the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the F X iron-sulfur cluster but that F X is not required for the biosynthesis of trimeric P700-A 1 cores. Since the PsaC protein requires the presence of F X for binding, the absence of F A and F B may be an indirect result of the absence of F X .

“…Transient EPR studies of the radical pair P ϩ A 1 Ϫ (1, 2) show that phylloquinone is oriented with its carbonyl bonds parallel to the vector joining P700 ϩ and A 1 Ϫ . Measurement of the dipolar coupling between the P700 ϩ and A 1 Ϫ radical pair yields the distance between these two radicals as 25.4 Ϯ 0.3 Å (3)(4)(5). A spin echo study of P700 ϩ A 1 Ϫ in single crystals of PS I (6) determined an angle of 27 Ϯ 5°between the dipolar axis between P700 ϩ and A 1 Ϫ and the crystallographic c axis (membrane normal).…”

Section: ؊ (253 ؎ 03 å) Is the Same As Between P700mentioning

confidence: 99%

“…However, without an accurate determination of the distance between the donor and acceptor, the data do not show unambiguously that the acceptor is bound at the A 1 site. Recently, ESE experiments have been used to determine the distance between P700 ϩ and A 1 Ϫ in native PS I (3)(4)(5) and to demonstrate that the distance remains the same for a number of nonnative quinones used in A 1 extraction/substitution experiments (4,29). The ESE of a weakly coupled spin correlated radical pair is phase shifted by 90°compared with that of a single radical and shows deep amplitude modulations as a function of the pulse spacing (30).…”

Section: Fig 4 Photoaccumulated and Simulated Q-band Cw Epr Spectramentioning

confidence: 99%

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

Zybailov¹,

Est²,

Zech³

et al. 2000

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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...