Biology subjects

Milrad, Y.

Publications and source records attributed to Milrad, Y..

7 recordsLinked to original sources

bioRxiv2025

Cryo-EM structure of Chlamydomonas Photosystem I complexedwith the alternative electron donor cytochrome c6

Photosynthetic electron transfer relies on small soluble carriers that shuttle electrons between the cytochrome bf complex and Photosystem I (PSI). While copper-containing plastocyanin (Pc) serves this role in plants, algae and cyanobacteria employ the heme protein cytochrome c (Cyt c) as well. Here we present a cryo-electron microscopy structure of a Cyt c:PSI complex from Chlamydomonas reinhardtii. The Cyt c heme is positioned [~]11 [A] from P700, stabilized by extensive contacts involving the N-terminal domain of PSAF. Importantly, R66 in Cyt c, a key residue in ancestral donors, forms a putative electrostatic contact with PsaB-D623 and participates in a tri-planar {pi}-stacking interaction with nearby aromatic residues. Our findings provide a structural framework for ancestral PSI interactions and illuminate the evolutionary diversification of electron transfer pathways.

biochemistry↗

bioRxiv2025

New Insights into Plastocyanin-Cytochrome b6f Formation: the Role of Plastocyanin Phosphorylation

In this work we investigated the role of plastocyanin (PC) phosphorylation in photosynthetic electron transfer, focusing on interactions with both cytochrome-b6f (Cytb6f) and Photosystem-I (PSI) in Chlamydomonas reinhardtii. While the binding and electron transfer between PC and PSI are well characterized, the interaction between PC and Cytf remains less clear. Using chemical cross-linking combined with mass spectrometry, we identified two potential binding models for PC and Cytf: "Side-on" and "Head-on". To evaluate electron transfer, we developed an in vitro system that allowed oxidized PC, formed via light-driven electron transfer at PSI, to re-oxidize Cytf. Our data shows that a phosphomimetic variant of PC, where phosphorylated PC S49 residue interacts with PetA-K188, displays faster Cytf oxidation, likely optimizing binding and electron transfer between PC and Cytf. Additionally, PC phosphomimetic variants exhibited slower transfer rates than wild type, suggesting that phosphorylation modulates PCs interaction with PSI. This regulation likely optimizes Cytf oxidation and electron transfer under conditions of low PC availability, such as during high light stress. Overall, PC phosphorylation appears to play a role in fine-tuning electron transfer between PSI, Cytb6f, and PC, thereby ensuring efficient photosynthesis in dynamic environmental conditions.

plant biology↗

bioRxiv2025

N-terminal region of PetD is essential for cytochrome b6f function and controls STT7 kinase activity via STT7-dependent feedback loop phosphorylation

The cytochrome b6f complex (b6f) links photosystem I (PSI) and photosystem II (PSII) in the photosynthetic electron transport chain and is distributed across appressed and non-appressed thylakoid membranes. The b6f also activates the state-transition 7 kinase (STT7), which phosphorylates light-harvesting complex proteins, triggering their migration to enable energy redistribution between PSII and PSI. STT7-dependent phosphorylation has also been observed at T4 in the N-terminal domain of the b6f subunit-IV (PetD), though its functional significance has remained unclear. To investigate its role, we generated several chloroplast mutants. The phosphomimic mutation PetD T4E - but not T4A - inhibited STT7 kinase activity, as indicated by the absence of STT7-dependent phosphorylation and a State 1-locked phenotype. This reveals a novel feedback mechanism regulating STT7-dependent phosphorylation. Deletion of the first five N-terminal amino acids similarly inhibited STT7 activity and additionally disrupted electron transfer, underscoring a crucial role of the PetD N-terminus in b6f function.

plant biology↗

bioRxiv2022

Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas reinhardtii with largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild type cells in comparison with crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnects redox control with active photosynthetic electron transport and metabolism as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding, that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning as well as for efficient photosynthesis. One-sentence summaryCalredoxin dependent redox regulation ensures efficient photosynthesis.

plant biology↗

bioRxiv2021

A novel PSII photosynthetic control is activated in anoxic cultures of green algae

Photosynthetic green algae face an ever-changing environment of fluctuating light as well as unstable oxygen levels, which via the production of free radicals constantly challenges the integrity of the photosynthetic complexes. To face such challenges, a complex photosynthetic control network monitors and tightly control the membrane redox potential. Here, we show that not only that the photosynthetic control set the rate limiting step of photosynthetic linear electron flow, but also, upon its ultimate dissipation, it triggers intrinsic alternations in the activity of the photosynthetic complexes. These changes have a grave and prolonged effect on the activity of photosystem II, leading to a massive 3-fold decrease in its electron output. We came into this conclusion via studying a variety of green algae species and applying advance mass-spectrometry and diverse spectroscopic techniques. Our results shed new light on the mechanism of photosynthetic regulation, and provide new target for improving photosynthesis.

plant biology↗

bioRxiv2020

Bi-directional electron transfer between H2 and NADPH mitigates the response to light fluctuations in green algae

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for interorganelle communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, in order to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still lacking. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and rely on indirect measurements. Here, we investigated this connection using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption is initially eventuated in H2 evolution, which initiate the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is recycled by Nda2 rather than consumed by terminal sinks such as CBB cycle and H2 production. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism. One sentence summaryEnergy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations.

plant biology↗

bioRxiv2020

The Mehler reaction site is the Phylloquinone within Photosystem I

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. Up to a third of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2*-), which occurs principally within photosystem I (PSI) via the Mehler reaction. Strikingly, the precise location as well as the evolutionary role of the reaction have long been a matter of debate. For decades, O2 reduction was assumed to take place solely in the distal iron-sulfur clusters of PSI rather than within the two asymmetrical cofactor branches. Here we demonstrate that under high irradiance, O2 photoreduction by PSI 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, and from the high rates of O2 photoreduction in PSI complexes lacking iron-sulfur clusters and in a mutant PSI, in which the lifetime of this phyllosemiquinone state is extended 100-fold. On these grounds, we suggest that the Mehler reaction serves as a release valve, functioning only when needed, under conditions where both the distal iron-sulfur clusters of PSI and the mobile ferredoxin pool are over reduced. SIGNIFICANCE STATEMENTPhotosynthesis is the process responsible for the oxygenation of the ancient anoxic atmosphere, and the transformation of inorganic carbon to most of the organic matter on Earth. However, it is less commonly appreciated that the appearance of oxygen in the atmosphere led to the unavoidable opposite process in which oxygen is consumed, thereby producing deleterious oxygen radicals such as the superoxide radical. For almost half a decade, the location of the main site of superoxide radical production in chloroplasts has been a matter of debate. We now provide conclusive evidence that it is located in the phylloquinones(s) within photosystem I.

plant biology↗