Near-IR absorbance changes and electrogenic reactions in the microsecond-to-second time domain in Photosystem I

Abstract

The back-reaction kinetics in Photosystem I (PS I) were studied on the microsecond-to-s time scale in cyanobacterial preparations, which differed in the number of iron-sulfur clusters to assess the contributions of particular components to the reduction of P700+. In membrane fragments and in trimeric P700-FA/FB complexes, the major contribution to the absorbance change at 820 nm (delta A820) was the back-reaction of FA- and/or FB- with lifetimes of approximately 10 and 80 ms (approximately 10% and 40% relative amplitude). The decay of photoinduced electric potential (delta psi) across a membrane with directionally incorporated P700-FA/FB complexes had similar kinetics. HgCl2-treated PS I complexes, which contain FA but no FB, retain both of these kinetic components, indicating that neither can be assigned uniquely to a specific acceptor. These results suggest that FA- reduces P700+ directly and argue for a rapid electron equilibration between FA and FB, which would eliminate their kinetic distinction in a back-reaction. In PsaC-depleted P700-Fx cores, as well as in P700-FA/FB complexes with chemically reduced FA and FB, the major contribution to the delta A820 and the delta psi decay is a biphasic back-reaction of F-X (approximately 400 microseconds and 1.5 ms) with some contribution from A-1 (approximately 10 microseconds and 100 microseconds), the latter of which is variable depending on experimental conditions. The delta A820 decay in a P700-A1 core devoid of all iron-sulfur clusters comprises two phases with lifetimes of 10 microseconds and 130 microseconds (2.7:1 ratio). The biexponential back-reaction kinetics found for each of the electron acceptors may be related to existence of different conformational states of the PS I complex. In all preparations studied, excitation at 532 nm with flash energies exceeding 10 mJ gives rise to formation of antenna 3Chl, which also contributes to delta A820 decay on the tens-of-microsecond time scale. A distinction between delta A820 components related to back-reactions and to 3Chl decay can be made by analysis of flash saturation dependencies and by measurements of kinetics with preoxidized P700.

The Cysteine-proximal Aspartates in the FX-binding Niche of Photosystem I

“…2 (A-G) shows ⌬A 826 kinetics of the DM-PS I complexes from the wild type and the aspartate mutants measured in the presence of the artificial electron donor, reduced DCPIP. As detailed previously (21,27), the slowest component in the wildtype DM-PS I complex ( Fig. 2A) has a lifetime of 2.2 s, and it arises from P 700 ϩ reduction by the external electron donor, ascorbate-reduced DCPIP.…”

“…In D557K PsaB , the component with a lifetime of ϳ15-20 s still had a relatively high contribution of 26.5% to the total absorbance change at flash intensities as low as 0.2 mJ, energies that produce a much lower (Ͻ10%) contribution of tens-ofmicroseconds components in the wild type (21). As shown in Fig.…”

“…At present, we cannot unambiguously ascertain whether this component represents P 700 ϩ reduction from an external donor or a back-reaction with endogenous acceptor. It should be noted, however, that kinetic components with lifetimes longer than 100 ms were not observed in absorbance changes in the near-IR (⌬A) and photovoltage (⌬ ) decay measurements performed in the absence of exogenous mediators (21). Faster components (Fig.…”

“…Room Temperature Transient Absorbance Spectroscopy-Transient absorbance changes of P 700 at 826 nm (⌬A 826 ) were measured in the microseconds to tens-of-seconds time domain with a laboratory-built double-beam spectrophotometer as described previously (21). Samples for optical experiments were suspended under anaerobic conditions at 50 g ml Ϫ1 Chl in 25 mM Tris buffer, pH 8.3, with 4 M DCPIP, 10 mM sodium ascorbate, and 0.03% DM.…”

“…PCC 6803, the major contribution to ⌬A 700 decay also shows biphasic kinetics with similar lifetimes of ϳ300 s and 1.05 ms; a point-by-point difference spectrum of the PsaC deletion mutant in the blue shows that the two kinetic phases are identical, with a bleaching from 400 to 500 nm and a broad peak at 430 nm characteristic of the weak S 3 Fe charge transfer bands of an iron-sulfur protein (19). In a P 700 -A 1 core devoid of F X , F B , and F A , the major contribution to ⌬A in the near-IR decays bi-exponentially with life times of 10 and 110 s (21,27). A point-by-point difference spectrum of the P 700 -A 1 core shows that the two kinetic phases are identical, with an absorbance increase from 340 to 410 nm characteristic of the 1-electron reduction of a quinone (27).…”