SummaryThe mechanisms of carbon concentration in marine diatoms are controversial. At low CO 2 , decreases in O 2 evolution after inhibition of phosphoenolpyruvate carboxylases (PEPCs), and increases in PEPC transcript abundances, have been interpreted as evidence for a C 4 mechanism in Thalassiosira pseudonana, but the ascertainment of which proteins are responsible for the subsequent decarboxylation and PEP regeneration steps has been elusive.We evaluated the responses of T. pseudonana to steady-state differences in CO 2 availability, as well as to transient shifts to low CO 2 , by integrated measurements of photosynthetic parameters, transcript abundances and quantitative proteomics.On shifts to low CO 2 , two PEPC transcript abundances increased and then declined on timescales consistent with recoveries of F v /F m , non-photochemical quenching (NPQ) and maximum chlorophyll a-specific carbon fixation (P max ), but transcripts for archetypical decarboxylation enzymes phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) did not change. Of 3688 protein abundances measured, 39 were up-regulated under low CO 2 , including both PEPCs and pyruvate carboxylase (PYC), whereas ME abundance did not change and PEPCK abundance declined.We propose a closed-loop biochemical model, whereby T. pseudonana produces and subsequently decarboxylates a C 4 acid via PEPC 2 and PYC, respectively, regenerates phosphoenolpyruvate (PEP) from pyruvate in a pyruvate phosphate dikinase-independent (but glycine decarboxylase (GDC)-dependent) manner, and recuperates photorespiratory CO 2 as oxaloacetate (OAA).
The desert microalga Chlorella ohadii was reported to grow at extreme light intensities with minimal photoinhibition, tolerate frequent de/re-hydrations, yet minimally employs antenna-based non-photochemical quenching for photoprotection. Here we investigate the molecular mechanisms by measuring Photosystem II charge separation yield (chlorophyll variable fluorescence, Fv/Fm) and flash-induced O yield to measure the contributions from both linear (PSII-LEF) and cyclic (PSII-CEF) electron flow within PSII. Cells grow increasingly faster at higher light intensities (μE/m/s) from low (20) to high (200) to extreme (2000) by escalating photoprotection via shifting from PSII-LEF to PSII-CEF. This shifts PSII charge separation from plastoquinone reduction (PSII-LEF) to plastoquinol oxidation (PSII-CEF), here postulated to enable proton gradient and ATP generation that powers photoprotection. Low light-grown cells have unusually small antennae (332 Chl/PSII), use mainly PSII-LEF (95%) and convert 40% of PSII charge separations into O (a high O quantum yield of 0.06mol/mol PSII/flash). High light-grown cells have smaller antenna and lower PSII-LEF (63%). Extreme light-grown cells have only 42 Chl/PSII (no LHCII antenna), minimal PSII-LEF (10%), and grow faster than any known phototroph (doubling time 1.3h). Adding a synthetic quinone in excess to supplement the PQ pool fully uncouples PSII-CEF from its natural regulation and produces maximum PSII-LEF. Upon dark adaptation PSII-LEF rapidly reverts to PSII-CEF, a transient protection mechanism to conserve water and minimize the cost of antenna biosynthesis. The capacity of the electron acceptor pool (plastoquinone pool), and the characteristic times for exchange of (PQH) with PQ and reoxidation of (PQH) were determined.
Crystals of photosystem II (PSII) contain the most homogeneous copies of the water-oxidizing reaction center where O 2 is evolved (WOC). However, few functional studies of PSII operation in crystals have been carried out, despite their widespread use in structural studies. Here we apply oximetric methods to determine the quantum efficiency and lifetimes of intermediates of the WOC cycle as a function of added electron acceptors (quinones and ferricyanide), both aerobically and anaerobically. PSII crystals exhibit the highest quantum yield of O 2 production yet observed of any native or isolated PSII (61.6%, theoretically 59 000 μmol O 2 /mg Chl/h). WOC cycling can be sustained for thousands of turnovers only using an electron acceptor (quinones, ferricyanide, etc.). Simulations of the catalytic cycle identify four distinct photochemical inefficiencies in both PSII crystals and dissolved PSII cores that are nearly the same magnitude. The exogenous acceptors equilibrate with the native plastoquinone acceptor at the Q B (or Q C ) site(s), for which two distinct redox couples are observable that regulate flux through PSII. Flux through the catalytic cycle of water oxidation is shown to be kinetically restricted by the Q A Q B two-electron gate. The lifetimes of the S2 and S3 states are greatly extended (especially S2) by electron acceptors and depend on their redox reversibility. PSII performance can be pushed in vitro far beyond what it is capable of in vivo. With careful use of precautions and monitoring of populations, PSII microcrystals enable the exploration of WOC intermediates and the mechanism of catalysis.
Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5-8× slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45°C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S=5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S=1/2 form.
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