Fast and global reorganization of the chloroplast protein biogenesis network during heat acclimation.

Photosynthesis is a central determinant of plant biomass production, but its homeostasis is increasingly challenged by heat. Little is known about the sensitive regulatory principles involved in heat acclimation that underly the biogenesis and repair of chloroplast-encoded core subunits of photosynthetic complexes. Employing time-resolved ribosome and transcript profiling together with selective ribosome proteomics, we systematically deciphered these processes in chloroplasts of Chlamydomonas reinhardtii. We revealed protein biosynthesis and altered translation elongation as central processes for heat acclimation and showed that these principles are conserved between the alga and the flowering plant Nicotiana tabacum. Short-term heat exposure resulted in specific translational repression of chlorophyll a-containing core antenna proteins of photosystems I and II. Furthermore, translocation of ribosome nascent chain complexes to thylakoid membranes was affected, as reflected by the increased accumulation of stromal cpSRP54-bound ribosomes. The successful recovery of synthesizing these proteins under prolonged acclimation of nonlethal heat conditions was associated with specific changes of the co-translational protein interaction network, including increased ribosome association of chlorophyll biogenesis enzymes and acclimation factors responsible for complex assembly. We hypothesize that co-translational cofactor binding and targeting might be bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.

Chloroplast ATP synthase biogenesis requires peripheral stalk subunits AtpF and ATPG and stabilization of atpE mRNA by OPR protein MDE1.

Chloroplast ATP synthase contains subunits of plastid and nuclear genetic origin. To investigate the coordinated biogenesis of this complex, we isolated novel ATP synthase mutants in the green alga Chlamydomonas reinhardtii by screening for high light sensitivity. We report here the characterization of mutants affecting the two peripheral stalk subunits b and b', encoded respectively by the atpF and ATPG genes, and of three independent mutants which identify the nuclear factor MDE1, required to stabilize the chloroplast-encoded atpE mRNA. Whole-genome sequencing revealed a transposon insertion in the 3'UTR of ATPG while mass spectrometry shows a small accumulation of functional ATP synthase in this knock-down ATPG mutant. In contrast, knock-out ATPG mutants, obtained by CRISPR-Cas9 gene editing, fully prevent ATP synthase function and accumulation, as also observed in an atpF frame-shift mutant. Crossing ATP synthase mutants with the ftsh1-1 mutant of the major thylakoid protease identifies AtpH as an FTSH substrate, and shows that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits. In mde1 mutants, the absence of atpE transcript fully prevents ATP synthase biogenesis and photosynthesis. Using chimeric atpE genes to rescue atpE transcript accumulation, we demonstrate that MDE1, a novel octotricopeptide repeat (OPR) protein, genetically targets the atpE 5'UTR. In the perspective of the primary endosymbiosis (~1.5 Gy), the recruitment of MDE1 to its atpE target exemplifies a nucleus/chloroplast interplay that evolved rather recently, in the ancestor of the CS clade of Chlorophyceae, ~300 My ago.

The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas (Chlamydomonas r einhardtii), chloroplast gene expression is tightly regulated posttranscriptionally by gene-specific trans-acting protein factors. Here, we report the identification of the octotricopeptide repeat protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase oligomycin-sensitive chloroplast coupling factor. Unlike most trans-acting factors characterized so far in Chlamydomonas, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5' untranslated regions of both the atpH and atpI genes. Coimmunoprecipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5' end of triphosphorylated atpH mRNA. A very similar sequence, located ∼60 nucleotides upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in Chlamydomonas. Such a dual-targeted trans-acting factor provides a means to coregulate the expression of the two proton hemi-channels.

Complexome profiling on the Chlamydomonas lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins.

While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.

MDA1, a nucleus-encoded factor involved in the stabilization and processing of the atpA transcript in the chloroplast of Chlamydomonas.

In Chlamydomonas reinhardtii, chloroplast gene expression is tightly regulated post-transcriptionally by gene-specific trans-acting protein factors. Here, we report the molecular identification of an OctotricoPeptide Repeat (OPR) protein, MDA1, which governs the maturation and accumulation of the atpA transcript, encoding subunit α of the chloroplast ATP synthase. As does TDA1, another OPR protein required for the translation of the atpA mRNA, MDA1 targets the atpA 5'-untranslated region (UTR). Unexpectedly, it binds within a region of approximately 100 nt in the middle of the atpA 5'-UTR, at variance with the stabilization factors characterized so far, which bind to the 5'-end of their target mRNA to protect it from 5' → 3' exonucleases. It binds the same region as TDA1, with which it forms a high-molecular-weight complex that also comprises the atpA mRNA. This complex dissociates upon translation, promoting degradation of the atpA mRNA. We suggest that atpA transcripts, once translated, enter the degradation pathway because they cannot reassemble with MDA1 and TDA1, which preferentially bind to de novo transcribed mRNAs.

Nitric Oxide Remodels the Photosynthetic Apparatus upon S-Starvation in Chlamydomonas reinhardtii.

Many photosynthetic autotrophs have evolved responses that adjust their metabolism to limitations in nutrient availability. Here we report a detailed characterization of the remodeling of photosynthesis upon sulfur starvation under heterotrophy and photo-autotrophy in the green alga (Chlamydomonas reinhardtii). Photosynthetic inactivation under low light and darkness is achieved through specific degradation of Rubisco and cytochrome b 6 f and occurs only in the presence of reduced carbon in the medium. The process is likely regulated by nitric oxide (NO), which is produced 24 h after the onset of starvation, as detected with NO-sensitive fluorescence probes visualized by fluorescence microscopy. We provide pharmacological evidence that intracellular NO levels govern this degradation pathway: the addition of a NO scavenger decreases the rate of cytochrome b 6 f and Rubisco degradation, whereas NO donors accelerate the degradation. Based on our analysis of the relative contribution of the different NO synthesis pathways, we conclude that the NO2-dependent nitrate reductase-independent pathway is crucial for NO production under sulfur starvation. Our data argue for an active role for NO in the remodeling of thylakoid protein complexes upon sulfur starvation.

Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism.

In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5' end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5' end by the cognate factor. Enzymatic removal of 5'-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32-34 nt ribosome-protected fragments. Using this 'in vivo ribosome footprinting', we identified the function and molecular target of two candidate trans-acting factors.

Spontaneous dominant mutations in chlamydomonas highlight ongoing evolution by gene diversification.

We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins.

Nitric oxide-triggered remodeling of chloroplast bioenergetics and thylakoid proteins upon nitrogen starvation in Chlamydomonas reinhardtii.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated.

A nucleus-encoded chloroplast protein regulated by iron availability governs expression of the photosystem I subunit PsaA in Chlamydomonas reinhardtii.

The biogenesis of the photosynthetic electron transfer chain in the thylakoid membranes requires the concerted expression of genes in the chloroplast and the nucleus. Chloroplast gene expression is subjected to anterograde control by a battery of nucleus-encoded proteins that are imported in the chloroplast, where they mostly intervene at posttranscriptional steps. Using a new genetic screen, we identify a nuclear mutant that is required for expression of the PsaA subunit of photosystem I (PSI) in the chloroplast of Chlamydomonas reinhardtii. This mutant is affected in the stability and translation of psaA messenger RNA. The corresponding gene, TRANSLATION OF psaA1 (TAA1), encodes a large protein with two domains that are thought to mediate RNA binding: an array of octatricopeptide repeats (OPR) and an RNA-binding domain abundant in apicomplexans (RAP) domain. We show that as expected for its function, TAA1 is localized in the chloroplast. It was previously shown that when mixotrophic cultures of C. reinhardtii (which use both photosynthesis and mitochondrial respiration for growth) are shifted to conditions of iron limitation, there is a strong decrease in the accumulation of PSI and that this is rapidly reversed when iron is resupplied. Under these conditions, TAA1 protein is also down-regulated through a posttranscriptional mechanism and rapidly reaccumulates when iron is restored. These observations reveal a concerted regulation of PSI and of TAA1 in response to iron availability.

The nucleus-encoded trans-acting factor MCA1 plays a critical role in the regulation of cytochrome f synthesis in Chlamydomonas chloroplasts.

Organelle gene expression is characterized by nucleus-encoded trans-acting factors that control posttranscriptional steps in a gene-specific manner. As a typical example, in Chlamydomonas reinhardtii, expression of the chloroplast petA gene encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on MCA1 and TCA1, required for the accumulation and translation of the petA mRNA. Here, we show that these two proteins associate in high molecular mass complexes that also contain the petA mRNA. We demonstrate that MCA1 is degraded upon interaction with unassembled cytochrome f that transiently accumulates during the biogenesis of the cytochrome b(6)f complex. Strikingly, this interaction relies on the very same residues that form the repressor motif involved in the Control by Epistasy of cytochrome f Synthesis (CES), a negative feedback mechanism that downregulates cytochrome f synthesis when its assembly within the cytochrome b(6)f complex is compromised. Based on these new findings, we present a revised picture for the CES regulation of petA mRNA translation that involves proteolysis of the translation enhancer MCA1, triggered by its interaction with unassembled cytochrome f.

PPR proteins of green algae.

Using the repeat finding algorithm FT-Rep, we have identified 154 pentatricopeptide repeat (PPR) proteins in nine fully sequenced genomes from green algae (with a total of 1201 repeats) and grouped them in 47 orthologous groups. All data are available in a database, PPRdb, accessible online at http://giavap-genomes.ibpc.fr/ppr. Based on phylogenetic trees generated from the repeats, we propose evolutionary scenarios for PPR proteins. Two PPRs are clearly conserved in the entire green lineage: MRL1 is a stabilization factor for the rbcL mRNA, while HCF152 binds in plants to the psbH-petB intergenic region. MCA1 (the stabilization factor for petA) and PPR7 (a short PPR also acting on chloroplast mRNAs) are conserved across the entire Chlorophyta. The other PPRs are clade-specific, with evidence for gene losses, duplications, and horizontal transfer. In some PPR proteins, an additional domain found at the C terminus provides clues as to possible functions. PPR19 and PPR26 possess a methyltransferase_4 domain suggesting involvement in RNA guanosine methylation. PPR18 contains a C-terminal CBS domain, similar to the CBSPPR1 protein found in nucleoids. PPR16, PPR29, PPR37, and PPR38 harbor a SmR (MutS-related) domain similar to that found in land plants pTAC2, GUN1, and SVR7. The PPR-cyclins PPR3, PPR4, and PPR6, in addition, contain a cyclin domain C-terminal to their SmR domain. PPR31 is an unusual PPR-cyclin containing at its N terminus an OctotricoPeptide Repeat (OPR) and a RAP domain. We consider the possibility that PPR proteins with a SmR domain can introduce single-stranded nicks in the plastid chromosome.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts.

We asked what peptide features govern targeting to the mitochondria versus the chloroplast, using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrion- and chloroplast-targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing lysines with arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.

Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts.

After endosymbiosis, organelles lost most of their initial genome. Moreover, expression of the few remaining genes became tightly controlled by the nucleus through trans-acting protein factors that are required for post-transcriptional expression (maturation/stability or translation) of a single (or a few) specific organelle target mRNA(s). Here, we characterize the nucleus-encoded TDA1 factor, which is specifically required for translation of the chloroplast atpA transcript that encodes subunit α of ATP synthase in Chlamydomonas reinhardtii. The sequence of TDA1 contains eight copies of a degenerate 38-residue motif, that we named octotrico peptide repeat (OPR), which has been previously described in a few other trans-acting factors targeted to the C. reinhardtii chloroplast. Interestingly, a proportion of the untranslated atpA transcripts are sequestered into high-density, non-polysomic, ribonucleoprotein complexes. Our results suggest that TDA1 has a dual function: (i) trapping a subset of untranslated atpA transcripts into non-polysomic complexes, and (ii) translational activation of these transcripts. We discuss these results in light of our previous observation that only a proportion of atpA transcripts are translated at any given time in the chloroplast of C. reinhardtii.

Light-driven processes: key players of the functional biodiversity in microalgae.

Microalgae are prominent aquatic organisms, responsible for about half of the photosynthetic activity on Earth. Over the past two decades, breakthroughs in genomics and ecosystem biology, as well as the development of genetic resources in model species, have redrawn the boundaries of our knowledge on the relevance of these microbes in global ecosystems. However, considering their vast biodiversity and complex evolutionary history, our comprehension of algal biology remains limited. As algae rely on light, both as their main source of energy and for information about their environment, we focus here on photosynthesis, photoperception, and chloroplast biogenesis in the green alga Chlamydomonas reinhardtii and marine diatoms. We describe how the studies of light-driven processes are key to assessing functional biodiversity in evolutionary distant microalgae. We also emphasize that integration of laboratory and environmental studies, and dialogues between different scientific communities are both timely and essential to understand the life of phototrophs in complex ecosystems and to properly assess the consequences of environmental changes on aquatic environments globally.

Les microalgues, organismes aquatiques majeurs, sont responsables de la moitié de l'activité photosynthétique planétaire. La lumière représente pour les microalgues une source d'énergie ainsi que d'informations sur leur environnement. Ces 20 dernières années, les progrès en génomique et biologie des écosystèmes et la disponibilité de ressources génétiques pour de nouvelles espèces modèles ont permis d'apprécier leur importance dans les écosystèmes globaux. Néanmoins, du fait de leur grande diversité et de leur histoire évolutive complexe, notre compréhension de la biologie des microalgues reste limitée. Nous nous concentrons ici sur la photosynthèse, la photoperception, et la biogenèse des plastes chez l'algue verte Chlamydomonas reinhardtii et les diatomées marines. Nous décrivons comment l'étude des processus gouvernés par la lumière ouvre de nouvelles perspectives pour l'étude de la biodiversité fonctionnelle des microalgues. Nous soulignons combien seule l'intégration d'études en laboratoire et en contexte environnemental et le dialogue entre les communautés scientifiques concernées permettront de comprendre la vie de ces phototrophes dans des écosystèmes complexes, et d'évaluer correctement les conséquences des changements environnementaux sur les milieux aquatiques.

An atypical haem in the cytochrome b(6)f complex.

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.

Evidence Supporting an Antimicrobial Origin of Targeting Peptides to Endosymbiotic Organelles.

Mitochondria and chloroplasts emerged from primary endosymbiosis. Most proteins of the endosymbiont were subsequently expressed in the nucleo-cytosol of the host and organelle-targeted via the acquisition of N-terminal presequences, whose evolutionary origin remains enigmatic. Using a quantitative assessment of their physico-chemical properties, we show that organelle targeting peptides, which are distinct from signal peptides targeting other subcellular compartments, group with a subset of antimicrobial peptides. We demonstrate that extant antimicrobial peptides target a fluorescent reporter to either the mitochondria or the chloroplast in the green alga Chlamydomonas reinhardtii and, conversely, that extant targeting peptides still display antimicrobial activity. Thus, we provide strong computational and functional evidence for an evolutionary link between organelle-targeting and antimicrobial peptides. Our results support the view that resistance of bacterial progenitors of organelles to the attack of host antimicrobial peptides has been instrumental in eukaryogenesis and in the emergence of photosynthetic eukaryotes.

The High Light Response and Redox Control of Thylakoid FtsH Protease in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the major protease involved in the maintenance of photosynthetic machinery in thylakoid membranes, the FtsH protease, mostly forms large hetero-oligomers (∼1 MDa) comprising FtsH1 and FtsH2 subunits, whatever the light intensity for growth. Upon high light exposure, the FtsH subunits display a shorter half-life, which is counterbalanced by an increase in FTSH1/2 mRNA levels, resulting in the modest upregulation of FtsH1/2 proteins. Furthermore, we found that high light increases the protease activity through a hitherto unnoticed redox-controlled reduction of intermolecular disulfide bridges. We isolated a Chlamydomonas FTSH1 promoter-deficient mutant, ftsh1-3, resulting from the insertion of a TOC1 transposon, in which the high light-induced upregulation of FTSH1 gene expression is largely lost. In ftsh1-3, the abundance of FtsH1 and FtsH2 proteins are loosely coupled (decreased by 70% and 30%, respectively) with no formation of large and stable homo-oligomers. Using strains exhibiting different accumulation levels of the FtsH1 subunit after complementation of ftsh1-3, we demonstrate that high light tolerance is tightly correlated with the abundance of the FtsH protease. Thus, the response of Chlamydomonas to light stress involves higher levels of FtsH1/2 subunits associated into large complexes with increased proteolytic activity.

Redesigning the QA binding site of Photosystem II allows reduction of exogenous quinones.

Strategies to harness photosynthesis from living organisms to generate electrical power have long been considered, yet efficiency remains low. Here, we aimed to reroute photosynthetic electron flow in photosynthetic organisms without compromising their phototrophic properties. We show that 2,6-dimethyl-p-benzoquinone (DMBQ) can be used as an electron mediator to assess the efficiency of mutations designed to engineer a novel electron donation pathway downstream of the primary electron acceptor QA of Photosystem (PS) II in the green alga Chlamydomonas reinhardtii. Through the use of structural prediction studies and a screen of site-directed PSII mutants we show that modifying the environment of the QA site increases the reduction rate of DMBQ. Truncating the C-terminus of the PsbT subunit protruding in the stroma provides evidence that shortening the distance between QA and DMBQ leads to sustained electron transfer to DMBQ, as confirmed by chronoamperometry, consistent with a bypass of the natural QA°- to QB pathway.