In vitro analysis of the plastid terminal oxidase in photosynthetic electron transport

Abstract

The plastid terminal oxidase PTOX catalyzes the oxidation of plastoquinol (PQH2) coupled with the reduction of oxygen to water. In vivo PTOX is attached to the thylakoid membrane. PTOX is important for plastid development and carotenoid biosynthesis, and its role in photosynthesis is controversially discussed. To analyze PTOX activity in photosynthetic electron transport recombinant purified PTOX fused to the maltose-binding protein was added to photosystem II-enriched membrane fragments. These membrane fragments contain the plastoquinone (PQ) pool as verified by thermoluminescence. Experimental evidence for PTOX oxidizing PQH2 is demonstrated by following chlorophyll fluorescence induction. Addition of PTOX to photosystem II-enriched membrane fragments led to a slower rise, a lower level of the maximal fluorescence and an acceleration of the fluorescence decay. This effect was only observed at low light intensities indicating that PTOX cannot compete efficiently with the reduction of the PQ pool by photosystem II at higher light intensities. PTOX attached tightly to the membranes since it was only partly removable by membrane washings. Divalent cations enhanced the effect of PTOX on chlorophyll fluorescence compared to NaCl most likely because they increase connectivity between photosystem II centers and the size of the PQ pool. Using single turnover flashes, it was shown that the level of reactive oxygen species, generated by PTOX in a side reaction, increased when the spacing between subsequent double flashes was enlarged. This shows that PTOX generates reactive oxygen species under limited substrate availability.

Distinguishing the roles of thylakoid respiratory terminal oxidases in the cyanobacterium Synechocystis sp. PCC 6803

“…However, since the PSI-less mutant is highly sensitive to light (Shen et al, 1993), the contribution of Cox as an electron shuttle to oxygen is likely to be less efficient compared with PSI activity, or this could be a transient phenomenon. It is possible that, under specific conditions, Cox also produces reactive oxygen species via a mechanism similar to PTOX in plants (Heyno et al, 2009;Feilke et al, 2014;Yu et al, 2014), thereby generating oxidative damage to the cells. On the other hand, rapid light curve analysis demonstrated a slight but significant difference in Y(ND) values between Δcyd and Δcox/cyd cells under higher light intensities, indicating competition between PSI and Cox for electrons in the Δcyd mutant (Fig.…”

Antioxidant and signaling functions of the plastoquinone pool in higher plants

“…When PTOX is overexpressed it can generate O 2 •− in the light (Heyno et al ), and such process was also shown in experiments with isolated enzyme (Feilke et al , Yu et al ). This may be the intrinsic feature of the enzyme, which performs the four‐electron reduction of the molecule O 2 through formation of the peroxide intermediates and can generate ROS in a side reaction with a rate that depends on the quinol concentration and pH value.…”

Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer

“…In the presence of DCMU, an inhibitor that binds to the Q B binding pocket, no difference between Cr‐PTOX1 and wild‐type was any longer observed (Figure a,b). Addition of the inhibitor octylgallate also abolished the difference between thylakoids from Cr‐PTOX1 plants and wild‐type (not shown, but see Feilke et al ., ).…”

“…We suppose that both, the pH value and the ion concentration, are crucial for the attachment of PTOX to the membrane. According to reconstitution experiments using recombinant Os‐PTOX fused with the maltose binding protein, a more alkaline pH and the presence of sodium in comparison to calcium increased the attachment of PTOX to the membrane (Feilke et al ., ). In the present state, we do not know if the association of PTOX with the membrane changes depending on the pH value or ion concentrations, if the interaction of PTOX with a putative partner protein is interrupted or if alterations of the pH induce a conformational shift in the PTOX protein structure that leads to a dissociation of the protein from the membrane.…”

“…Cr‐PTOX1 plants produced more O 2 •− /H 2 O 2 (Figure S3, Heyno et al ., ) indicating that PTOX acts in a pro‐oxidant manner. Depending on the plastoquinol concentration, PTOX, besides catalyzing the complete reduction of O 2 to H 2 O, generates partially ROS as has been demonstrated in vitro using recombinant PTOX from rice (Feilke et al ., ; Yu et al ., ). Transformants with increased PTOX levels have been shown to exhibit either no phenotype under photoinhibitory conditions (Rosso et al ., ) or to exhibit signs of photoinhibition (Heyno et al ., ; Ahmad et al ., ).…”