Redox-potentiometric titrations of the electrochromic absorption change in chloroplasts

Abstract

Two phases of the electrochromic 515 nm absorption change in chloroplasts elicited by microsecond flashes can be resolved kinetically. Redox-potentiometric titrations indicate that the initial amplitude appearing within 0.5 ms, and designated as phase a, has three components in the low-potential region with E m7.5 values of +60 mV, −195 mV and less than −400 mV. From the insensitivity to DCMU, we propose that the species with E m7.5 values of −195 mV and less than −400 mV are both related to Photosystem I. This conclusion was supported by the loss of both components when the Photosystem I reaction centre (P-700) was chemically oxidised ( E m7.5 = +370 mV). The species having an E m7.5 less than −400 mV is presumed to be the Photosystem I primary acceptor, while the E m7.5 = −195 mV wave could be due to a secondary electron acceptor, such as cytochrome b-563 LP, whose photoreduction is possible owing to the long duration of the excitation flash. The DCMU-sensitive component with an E m7.5 of +60 mV is assumed to be the primary quinone acceptor (Q A) of Photosystem II. Unlike the Photosystem I redox components, the midpoint potential of this species is sensitive to the background ionic level: the E m7.5 is shifted to −100 mV when the cation concentration is lowered to facilitate membrane unstacking. The slow phase of the electrochromic signal (phase b) has been estimated by measuring the 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone-sensitive amplitude of the absorption change at 20 ms. The signal appears with an estimated E m7.5 = +50 mV, becomes maximal at −50 mV and attenuates with an E m7.5 of about −180 mV. These results suggest that phase b occurs when the plastoquinone pool is reduced and cytochrome b-563 LP is oxidised.