HPLC determination of chlorophyll a' and phylloquinone in photosystem I of higher plants and cyanobacteria

Abstract

Stoichiometries of Chlorophyll (Chl) a¿, C132-epimer of Chl a, within Photosystem (PS) I of higher plants and cyanobacteria were determined by simultaneous detection of Chl a¿ and phylloquinone, the secondary electron acceptor of PS I (two molecules per PS I), with reversed-phase HPLC analyses, and spectrophotometric determination of P700, the primary donor of PS I. For thermophilic cyanobacterium Synechococcus elongatus, the Chl a¿/PhQ ratio was about 0.5 for cell, thylakoid membranes, and PS I trimer. The Chl a¿/P700 ratio and the PhQ/P700 ratio were about 1 and 2, respectively, for thylakoid membranes and PS I trimer. Theses results showed that a 1:1:2 stoichiometry is established among Chl a¿, P700, and PhQ within PS I of S. elongatus. PS I trimer isolated from Synechocystis PCC 6803 also showed the same stoichiometry as that found for S. elongatus. Native PS I complexes of spinach and barley also showed the 1:1:2 stoichiometry among Chl a¿, P700, and PhQ. Treatments of PS I with water-saturated/dry diethyl ether mixtures enriched Chl a¿ up to the Chl a/Chl a¿ ratio of 9-10 with keeping the Chl a¿/P700 ratio at about 1. These findings show that higher plant PS I contains one molecule of Chl a¿ in close association with the primary donor, P700. Pigment composition analyses with reversed-phase HPLC indicate that one molecule of Chl a¿ is universally present in PS I of oxygenic photo-autotroph as the indispensable ingredient, possibly as the constituent of the primary donor, P700.