Pigment Quantitation and Analysis by HPLC Reverse Phase Chromatography:  A Characterization of Antenna Size in Oxygen-Evolving Photosystem II Preparations from Cyanobacteria and Plants

Patzlaff, Jason S.; Barry, Bridgette A.

doi:10.1021/bi960056z

Cited by 39 publications

(65 citation statements)

References 53 publications

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“…The presence of CP43Ј may be a primary reason why certain pigments were found to be present at higher levels than expected. Biochemical analyses sometimes gave rise to higher pigment contents, e.g., 40-55 Chl (21,22) than are observable in the crystal structure; this tendency is more pronounced for lipids (23).…”

Section: Resultsmentioning

confidence: 99%

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Identification of the special pair of photosystem II in a chlorophyll d -dominated cyanobacterium

Tomo

Okubo

Akimoto

et al. 2007

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

125

127

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The composition of photosystem II (PSII) in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43 (PcbC), D1, D2, cytochrome b 559, PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 ؎ 7 Chl d, 3.0 ؎ 0.4 Chl a, 17 ؎ 3 ␣-carotene, and 1.4 ؎ 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina, our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system. Acaryochloris marina ͉ FTIR ͉ reaction center ͉ photosynthesis ͉ electron transfer C hlorophyll (Chl) a has a ubiquitous role as an electron donor in the photochemical reactions of oxygenic photosynthesis, in which two kinds of photosystem, namely photosystem I (PSI) and photosystem II (PSII), cooperatively drive photosynthetic electron flow from water to NADP ϩ . The reduced cofactor, NADPH, is then used for CO 2 fixation. Chl a is a key pigment that serves as an electron donor called the special pair in PSI and PSII. Acaryochloris spp. are unique cyanobacteria that differ from the majority of photosynthetic organisms by having Chl d (3-desvinyl-3-formyl Chl a) (1-4) as the major pigment (Ͼ95%); Chl a is a minor component but is never absent (5, 6). In photosynthetic organisms, changes in pigment composition affect both the function of pigments and their reaction environments, including the modified proteins that accommodate them. Photosynthetic pigments function in two roles: as lightharvesting components and as electron transfer components. Light harvesting is mainly governed by the orientation and energy levels of pigments, and, in this context, a particular excitation energy level is not an absolute precondition for function, but a relative one. On the other hand, electron transfer reactions are governed by an absolute redox potential, because the photosynthetic oxidation of water requires a very high potential, whereas reduction of NADP ϩ requires a very low potential. For this reason, it is of particul...

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Section: Resultsmentioning

confidence: 99%

“…We used published molar extinction coefficients for the individual pigments (45)(46)(47). The PQ-9 content was measured by reverse-phase HPLC as described in detail elsewhere (21).…”

Section: Methodsmentioning

confidence: 99%

Identification of the special pair of photosystem II in a chlorophyll d -dominated cyanobacterium

Tomo

Okubo

Akimoto

et al. 2007

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

125

127

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“…Formation of the phenylhydrazone during phenylhydrazine titration of AO has been monitored by an increase in absorbance at 450 nm (46). However, monitoring of phenylhydrazine binding to PSII by this approach is not possible due to spectral interference from pigments associated with PSII (47,48).…”

Section: Discussionmentioning

confidence: 99%

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Anderson

Ouellette

Barry

2000

Journal of Biological Chemistry

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-[14 C]butylamine and phenylhydrazine were employed as probes. Neither compound can be oxidized by a transamination or addition/elimination mechanism, but both can react with activated carbonyl groups, produced as a result of posttranslational modification of amino acid residues, to give amine-derived adducts.14 C incorporation into the PSII subunits D2/D1 and CP47 was obtained upon treatment of PSII with either t-[ 14 C]butylamine or [ 14 C]phenylhydrazine. For t-butylamine and methylamine, the amount of labeling increased when PSII was treated with denaturing agents. Labeling of CP47, D2, and D1 with methylamine and phenylhydrazine approached a one-to-one stoichiometry, assuming that D2 and D1 each have one binding site. Evidence was obtained suggesting that reductive stabilization and/or access are modulated by PSII light reactions. These results support the proposal that PSII subunits D2, D1, and CP47 contain quinocofactors and that access to these sites is sterically limited.Photosystem II (PSII), 1 a membrane-bound protein complex, catalyzes the light-driven oxidation of water and the reduction of plastoquinone. PSII consists of hydrophobic and extrinsic subunits and contains chlorophyll, plastoquinone, manganese, and several other bound cofactors. Water oxidation occurs at a manganese-containing catalytic site located on the lumenal side of the membrane. After photoexcitation, the primary chlorophyll donor, P 680 , transfers an electron from its excited state to a pheophytin molecule. Pheophytin transfers the electron to a quinone acceptor molecule, Q A , which in turn reduces a second quinone acceptor, Q B (reviewed in Refs. 1 and 2). PSII contains two well characterized redox-active tyrosines, Z and D (3, 4). Z, an intermediate electron carrier between P 680 and the manganese cluster, reduces oxidized P 680 (5). D forms a stable radical and has no known role in water oxidation (6). Oxidation of a third, posttranslationally modified tyrosine, M, occurs in PSII site-directed mutants (6 -9).Primary amines are known to be substrate analogs and inhibitors of oxygen evolution (10 -12). Recently, the covalent incorporation of 14 C-labeled primary amines into PSII subunits has been observed under reducing conditions (13). Evidence for oxidation of [ 14 C]benzylamine by PSII was obtained by detection and quantitation of [ 14 C]benzaldehyde (13). Because PSII exhibits this amine oxidation activity and an aldehyde product has been observed, covalent incorporation of 14 C from labeled primary amines into PSII subunits could be caused by two possible reactions (13). In the first possible reaction, addition of reductant traps a Schiff base complex of substrate ( 14 C-labeled amine) and an amino acid side chain that has been posttranslationally modified to contain an activated carbonyl group (13,14,15). In the second possible reaction, addition of reductant traps a Schiff base complex of product ( 14 C-labeled aldehyde) and the amino group of a lysine side chain (13). When formaldehyde is produced, this reaction produ...

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“…[18][19][20] Since Chl a is known to undergo several alterations in methanol, 13 we sought for an eluent containing a minimal amount of methanol. After examination of several mixtures of acetonitrile, alcohols and water, the eluent described in Experimental gave satisfactory resolution for PhQ, Chl a and Chl a′.…”

Section: Separation and Identification Of Photosynthetic Pigmentsmentioning

confidence: 99%

Separation and Determination of Minor Photosynthetic Pigments by Reversed-phase HPLC with Minimal Alteration of Chlorophylls

Nakamura

Watanabe

2001

ANAL. SCI.

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Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a′ (the C13 2 -epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS I), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a′ nor Pheo a, showed the Chl a′/Chl a ratio of 3 -4 × 10 -4 and the Pheo a/Chl a ratio of 4 -5 × 10 -4 , showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a′ ratio of 0.58 ± 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a′ per PS I.

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Pigment Quantitation and Analysis by HPLC Reverse Phase Chromatography: A Characterization of Antenna Size in Oxygen-Evolving Photosystem II Preparations from Cyanobacteria and Plants

Cited by 39 publications

References 53 publications

Identification of the special pair of photosystem II in a chlorophyll d -dominated cyanobacterium

Identification of the special pair of photosystem II in a chlorophyll d -dominated cyanobacterium

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Separation and Determination of Minor Photosynthetic Pigments by Reversed-phase HPLC with Minimal Alteration of Chlorophylls

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