Reversed-phase HPLC Determination of Chlorophyll a′ and Naphthoquinones in Photosystem I of Red Algae: Existence of Two Menaquinone-4 Molecules in Photosystem I of Cyanidium caldarium

Abstract

The light reaction of oxygenic photosynthetic organisms proceeds by the cooperation of hundreds of such cofactors as chlorophyll (Chl), quinones, and carotenoids, placed within large pigment-protein complexes, photosystem (PS) I and PS II, in thylakoid membranes. Most Chl molecules function as lightharvesting pigments to funnel the excitation energy to the primary electron donor, P700 in PS I and P680 in PS II (for review, see Ref. 1). In the electron-transfer chains of PS I and PS II, minor Chl a derivatives play crucial roles in light-induced charge separation. 2 PS II contains two molecules of demetallated Chl a, pheophytin (Phe) a, as the primary electron acceptor. 3 We have proposed the existence of one or two molecules of Chl a′, the C13 2 -epimer of Chl a, in PS I through HPLC analysis of pigments.4,5 Recent X-ray crystallography on the PS I trimer of a thermophilic cyanobacterium Synechococcus elongatus revealed that P700 is a heterodimer of Chl a′ and Chl a. However, whether other oxygenic photosynthetic organisms use one Chl a′ molecule in P700 is still unknown. Because Chl a′ has practically the same absorption and fluorescence spectra as Chl a, 7 but shows a different chromatographic behavior, 8 an HPLC analysis of pigment extracts is the sole reliable way for the precise determination of Chl a′ in PS I. For this purpose, the conditions for pigment extraction and HPLC must strictly ensure the molecular integrity of Chl a, since Chl a undergoes several alterations in organic solvents. 9 Recently, we developed such conditions which can deduce the Chl a′/PS I ratio on a single reversed-phase HPLC trace by the simultaneous detection of Chl a′ and phylloquinone (PhQ), the secondary electron acceptor of PS I. 10 Pigment composition analyses of two cyanobacteria, a green alga Chlamydomonas reinhardtii, and spinach, combined with spectrophotometric determination of the Chl a/P700 ratios showed that one Chl a′ molecule exists in the vicinity of P700 in these organisms. 38 The light-harvesting system exhibits a significant variety among oxygenic photosynthetic organisms. 11 Typical cyanobacteria use soluble membrane-extrinsic phycobiliproteins as a light-harvesting system. Green algae and higher plants utilize only membrane-intrinsic Chl a/b complexes, LHC I and LHC II. The light-harvesting systems of red algae are known to be a transitional state from cyanobacteria to green plants, 12,13 and contain both phycobiliprotein and membrane-intrinsic LHC I (containing Chl a alone as the chlorophyllous pigment). 14,15 Hence, the pigment composition of red algae is of much interest to examine the universality of the existence of one Chl a′ molecule in P700.In view of this, we extended the pigment composition analysis to two red algae, a marine alga Porphyridium purpureum employed for many biochemical studies [12][13][14] and an alga living in an extreme environment, Cyanidium caldarium. 16 Experimental Photosynthetic organismsPorphyridium purpureum IAM R-1 was grown in an artificial seawater medium 17 for 7 days at 25...

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

“…PCC 7002 were shown to contain MQ-4 instead of PhQ, the quinone present in most other cyanobacteria and higher plants. MQ-4 was recently shown to be present in the red alga C. caldarium (2) and the early diverging cyanobacterium, Gloeobacter violaceus PCC 7421 (37). These results indicate that MQ-4 may be more widely employed in PS I than was previously recognized.…”

“…In most organisms, including Synechocystis sp. PCC 6803, the quinone in the A 1 site is phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), but in Euglena gracilis and Anacystis nidulans, the quinone is 5Ј-monohydroxyphylloquinone (1), and in the red alga Cyanidium caldarium it is menaquinone-4 (MQ-4) 1 (2). Our approach to studying structural and functional relationships involving A 1 is to replace the native quinone in PS I with quinones that have different thermodynamic and structural properties but are still able to mediate electron transfer from A 0 to the Fe/S clusters.…”

A Fluorescent-Based HPLC Assay Using 4-Chloro-7-nitrobenzo-2-oxa-1, 3-diazole as Derivatization Agent for the Determination of Iron Bioavailability to Red Tide Phytoplankton

“…Specific HPLC chromatogram peaks of known proteins or the ratio of two protein peaks (e.g. flavodoxin and ferredoxin) has been used for the determination of the nutritional status of seawaters [16,18,27]. For example, Erdner and Anderson [18] …”

“…Fluorescent-based HPLC assay is a quick, easy, efficient and powerful method for the quantitative measurement of cellular macromolecules in organisms [22][23][24][25][26][27]. This method has been used for the determination of cysteine and cysteamine adducts in Escherichia coli-derived proteins [26], thiol compounds in biological samples [22], human erythrocytic glutathione [25], domoic acid (DA) in mussels (Mytilus edulis) [24], peptides in tissue and plasma samples [28] and amines and proteins [29] using different fluorescent reagents as the derivatizer.…”

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

“…4b) species are present and function as A 1 in their RCs. Kobayashi et al (2006a) As mentioned above, A 1 in oxygenic PS I is PhQ, however, MQ was recently reported to function as A 1 in a primitive unicellular red alga Cyanidium caldarium (Yoshida et al 2003), then in a primitive cyanobacterium G. violaceus , and a marine centric diatom Chaetoceros gracilis (Ikeda et al 2008). It has been shown that A. marina uses PhQ as A 1 (Ohashi et al 2008a).…”