Phylloquinone is the principal Mehler reaction site within photosystem I in high light

Abstract

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•-) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron-sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron-sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron-sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

Figure 1

Diagram of electron transfer in PSI with lifetimes and mid-point redox potentials (E_m) of cofactors. A, For the WT according to Ptushenko et al. (2008). B, For PsaA-F₆₈₉N mutant taking into account for the effect of mutation on the E_m of PhQ_A/${\text{PhQ}}_{\mathrm{A}}^{•-}$ according to Santabarbara et al. (2015); blue lines represent E_m of O₂/${\mathrm{O}}_2^{•-}$ in water (−160 mV) and dimethylformamide (−500 to −600 mV; Wardman, 1990). P₇₀₀, the dimer of Chl a molecules in PSI; A₀, the primary electron acceptor in PSI; PhQ, phylloquinone, a secondary electron acceptor in PSI; F_X, a 4Fe–4S cluster, a secondary electron acceptor in PSI; F_A and F_B, 4Fe–4S clusters, the terminal electron acceptors in PSI; Fd, ferredoxin, the mobile electron acceptor. Modified from Kozuleva and Ivanov (2016).

Figure 2

Effect of Pc concentration on the O₂ photoreduction rate in the absence or the presence of MV. Mature WT PSI complexes from C. reinhardtii, 15 nM; Asc, 10 mM; initial O₂ concentration, 250 µM; irradiance, 2,200 µmol photons m⁻² s⁻¹; where indicated MV, 50 µM. Values are mean for three to four replicates; ±se.

Figure 3

Effect of irradiance on the apparent rate constant of O₂ photoreduction (k₂) by fully mature intact PSI complexes from WT strain of C. reinhardtii. A, In the absence of MV; the rate of O₂ consumption observed at atmospheric pressure of O₂ (dashed line) is shown for comparison. B, In the presence of MV at 50 µM (dark blue line, closed circles) or at 4 µM (light blue line, open circles); the curve observed in the absence of MV from (A) (green line, closed squares) is shown for comparison. Mature WT PSI complexes, 10 nM; Asc, 10 mM; catalase, 500 U mL⁻¹; Pc, 5 µM; initial O₂ concentration, 10–30 µM. Values are mean for 7–10 replicates; ±se.

Figure 4

Effect of irradiance on the apparent rate constant of O₂ photoreduction (k₂) by PSI preparations from C. reinhardtii challenging PhQs contribution to O₂ photoreduction. A, F_X- and A₁-core complexes; B, The PsaA-F₆₈₉N mutant; the curve observed for intact WT PSI complexes in the absence of MV from Figure 3A (green line, closed squares) is shown on both panels for comparison. The F_X-core, A₁-core, PSI^AFN complexes, 10 nM; TMPD, 1 mM (A), or Pc, 5 µM (B); Asc, 10 mM; catalase, 500 U mL⁻¹; initial O₂ concentration, 10–30 µM. Values are mean for 7–10 replicates; ±se.

Figure 5

Effect of irradiance on the apparent rate constant of O₂ photoreduction (k₂) by fully mature WT PSI complexes from C. reinhardtii concomitantly reducing NADP⁺. A, Effect of irradiance on apparent k₂ in the presence of Fd (orange triangles) or Fd, FNR, NADP⁺ (purple circles); the curve observed for intact WT PSI complexes in the absence of Fd from Figure 3A (green squares) is shown for comparison; B, Effect of irradiance on the rate of NADP⁺ reduction. Mature WT PSI complexes, 10 nM; Pc, 5 µM; Asc, 10 mM; catalase, 500 U mL⁻¹; Fd, 5 µM; where indicated: FNR, 200 nM, and NADP⁺, 1 mM; initial O₂ concentration, 10–30 µM. Values are mean for 7–10 replicates for k₂ and five replicates for rates; ±se.