Chlorophyll is the world’s most abundant pigment and essential for oxygenic photosynthesis. It is assembled in the pigment-binding proteins of the core and light-harvesting complexes of photosystems I and II (PSI and PSII) and absorbs light for the downstream energy conversion through the photosynthetic electron transport chain. The final steps of chlorophyll synthesis in angiosperms require a tight and light-dependent control due to the exclusively light-operating protochlorophyllide oxidoreductase (POR). Moreover, the synchronized synthesis of chlorophyll and nuclear and plastid-encoded chlorophyll-binding proteins ensures the supply of stoichiometric amounts of chlorophyll for the direct association with the pigment-binding proteins. As an additional level of regulation, posttranslational control mechanisms of enzymes in chlorophyll biosynthesis allow a rapid response to the continuously changing environmental conditions, for example, the daily varying light intensities and temperatures.

The redox-dependent control of proteins is based on the thiol-disulfide changes of cysteine residues (Cys) and belongs to the stringent posttranslational regulatory mechanisms, which maintain stability, catalytic activity or protein-protein interactions in multiple cellular processes, including the metabolic pathway of chlorophyll biosynthesis (Wittmann et al., 2020). Photosynthetic electron transport provides a reducing environment in chloroplasts, making electrons available for many metabolic pathways. Reduced ferredoxin provides electrons for the thioredoxin (TRX)-driven redox machinery via ferredoxin-thioredoxin reductase (FTR; Buchanan and Balmer, 2005; Dai et al., 2007)., Among others in plastids, electrons can also be transferred through the NADPH-dependent thioredoxin reductase C (NTRC) to 2-cysteine peroxiredoxin (2-CP), to detoxify also hydrogen peroxide (H₂O₂, Serrato et al., 2004; Toivola et al., 2013; Pérez-Ruiz et al., 2017; Cejudo et al., 2019). It was previously thought that NTRC also contribute to the reduction of enzymes of chloroplastic primary metabolism (Moon et al., 2006; Thormählen et al., 2013; Pérez-Ruiz et al., 2014). The more recent proposal by Perez-Ruiz et al. is based on the analysis of ntrc mutants, according to which the altered redox states of plastidic enzymes are not primarily explained by the absence of NTRC, but by the lack of reducing power of TRX isoforms, which complementarily substitute the absence of NTRC for the reduction of 2-CP, thus neglecting the enzymes that are the actual TRX-dependent targets (Perez-Ruiz et al., 2017).

Among the TBS enzymes, 5-aminolevulinic acid dehydratase (ALAD), also known as porphobilinogen synthase (PBGS), was identified as an interaction partner of NTRC, f- and m-type TRXs by pulldown and bimolecular fluorescence complementation (BiFC) assays (Wittmann et al., 2018). These findings confirmed previous results, which were initially obtained through different proteomic screens of protein extracts from Synechocystis PCC6803 (Lindahl and Florencio, 2003) and Chlamydomonas reinhardtii (Pérez-Pérez et al., 2017) and Arabidopsis thaliana (González et al., 2019) suggesting ALAD as an interaction partner of these plastidic reductants

The two paralogous HEMB1 (AT1G69740) and HEMB2 (AT1G44318) genes encode for the isoforms ALAD1 and ALAD2 in A. thaliana, respectively. ALAD1 is the dominant, possibly exclusive, variant in leaves as reported in Tang et al. (2012). Due to its essential importance, ALAD1 was used in our studies and for simplicity we refer to this isoform as ALAD throughout the manuscript. ALAD catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to porphobilinogen (PBG). ALA is formed in the rate-limiting step of TBS by glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSAAT) (Tanaka and Tanaka, 2007). While the ALAD orthologues from human, animals, fungi, archaea and most bacteria recruit Zn²⁺-ions in their catalytic center (Jaffe, 2016), plant ALAD binds up to three Mg²⁺ cations, which are suggested to act (i) for catalysis in the active center, (ii) as an allosteric activator of the transition between different oligomeric ALAD forms and (iii) as an inhibitor during high Mg²⁺ concentration (Kervinen et al., 2000; Kokona et al., 2008; Jaffe, 2016). Interestingly, the studies of the posttranslational control of ALAD activity revealed a unique mechanism in TBS. Human and pea ALAD have been suggested to balance a regulatory equilibrium between a homohexameric, inactive form and a homooctameric, active form via the dissociation into dimeric intermediates. Thereby, ALAD interconverts between a “detached” and a “hugging” dimer (Kokona et al., 2008).

The plant ALAD activity depends on the presence of Mg²⁺ and differs pH-dependently with an enzymatic optimum in a slightly alkaline environment (Balange and Lambert, 1983; Kervinen et al., 2000). These two features considerably vary in chloroplasts during the diurnal plant growth. Moreover, ALAD activity is promoted by reducing agents, such as dithiothreitol (DTT) and the two isoforms TRX-f and TRX-m (Balange and Lambert, 1983). TRX-f turned out to be the more effective activator of ALAD than TRX-m (Balange and Lambert, 1983).

By a virus-induced gene silencing (VIGS) approach, the expression of the Arabidopsis TRXm2, TRXm4 and TRXm1 genes was simultaneously inactivated and the content of ALAD was decreased in the transgenic Arabidopsis lines compared to WT controls (Da et al., 2017). Similarly, the ntrc and trxf1 seedlings contain less ALAD than wild type (WT), but not as much decreased as in TRX-m deficient plants (Wittmann et al., 2018). The lower content of ALAD correlates with its decreased in vivo activity in leaf extracts (Wittmann et al., 2018). As TRX also stimulates in vitro ALAD activity, it is intended to examine whether thiol-based redox regulation by TRX not only affects the stability of ALAD, but also its enzymatic activity. In continuation to our previous investigations on the redox control in TBS, we aimed to elucidate the possible thiol switches at the redox-sensitive Cys residues of A. thaliana ALAD and to identify the potential Cys residues for the thiol-based redox control. By using recombinant WT and Cys substitution mutants of ALAD their feasible redox switches were explored.

Crystal structures of the Mg²⁺-dependent ALAD have been published from the gram-negative rod-shaped bacterium Pseudomonas aeruginosa (Frankenberg et al., 1999; Frere et al., 2002), and the photosynthetically active green sulfur bacterium Chlorobium vibrioforme (Coates et al., 2004; Coates et al., 2005). Both structures are strongly conserved. Based on these structures we modeled the A. thaliana ALAD (AtALAD) structure by means of AlphaFold [ Figure 1 , (Jumper et al., 2021)]. ALAD1 consists of 430 amino acid residues (including a 52 amino acid residue-long N-terminal, plastid transit peptide; from here on we call this isoform exclusively ALAD) and contains six Cys, whereby four of these are conserved in higher plants (Cys152, Cys251, Cys404, and Cys426, Figure 1 ). The monomeric structure of the mature ALAD comprises a flexible N-terminal arm and a globular peptide with a αβ-barrel domain, which include the active site between D220 and Y416 of AtALAD (predicted by Conserved Domain Database; Lu et al., 2020). It is worth noting that the variable N-terminal arm of the human was proposed to be decisive for the dynamic formation of the hugging and the detached dimer prior to the structural conversion into octameric and hexameric configuration (Breinig et al., 2003; Jaffe, 2016). The estimation of the probability of the modeled structure of ALAD is less predictable for some peptide domains due to their greater dynamic flexibility ( Figure 1 ). This prediction-based analysis of the ALAD structure revealed that pLDDT values are low at the N-terminus, C-terminus and in the loop between Gly305 and Glu319, indicating a higher structural instability of these regions. These values also indicate the importance of the variable conformations in these regions for enzyme activity, oligomerisation and stability (Guo et al., 2022). The long mobile region of the N-terminus exhibits high flexibility and can swing around within a radius of about 40-60 Å, while the flexibility of the Gly305-Glu319 peptide region may be related to the entry and exit of Mg²⁺ ions and the ligands ALAD.

Based on the crystal structures of the two ALADs from P. aeruginosa and C. vibrioforme, we also modelled the structure of the dimeric ALAD from Arabidopsis. The three aligned structures showed a high degree of conformational similarity, with the exception of the N-terminal arm of the plant ALAD. Interestingly, the N-terminal arm of one ALAD molecule apparently interacts with the globule structure of the second monomer ( Figure 1 ).

Plant TBS is light-dependently stimulated, while darkness suppresses the initial metabolic step, the synthesis of ALA, in response to the inactive light-operating POR (Richter et al., 2010). Thus, the metabolic flow between ALA synthesis and protochlorophyllide reduction is tightly regulated during day- and nighttime, but also balanced in response to varying environmental conditions (Brzezowski et al., 2015; Hou et al., 2019; Richter et al., 2019). An adjustment to the changing growth requirements is expected at almost every catalytic step, including ALAD, to obtain an instantaneous balance of proteostasis. This includes activity, stability, interaction with neighbor enzymes or oligomerisation and aggregation of the participating proteins. Redox-dependent control of ALAD activity and stability is the proposed mechanism at the step of PBG synthesis. Photosynthesis ensures an adequate environment of reducing conditions in the chloroplast, which may maintain ALAD active and stable. Light-induced transcriptional control of the HEMB1 gene (Tang et al., 2012) is likely not involved in the rapid fine-tuning of ALAD activity.

It is interesting that native Arabidopsis extracts contain ALAD in protein complexes of different molecular sizes. AtALAD was found not only as a monomer and dimer, but also as at least two multimeric variants after separation in BN-polyacrylamide gels. Moreover, dimers were also disclosed in reducing and denatured SDS gels ( Figure 4 ) and could be confirmed by modelling the ALAD structure based on the crystal structure of the two Mg-containing bacterial ALAD from P. aeruginosa and C. vibrioforme ( Figure 1 ). In consistency, recombinant pea ALAD migrated also as two different multimers in non-denaturing gels. And it has been shown for human, bacterial, and pea ALAD that these homologous proteins are similar in their ability to form equilibrium between an octamer and a hexamer, with both oligomers formed via the formation of two distinct intermediate structures of dimers (Tang et al., 2006; Lawrence et al., 2009). As the recombinant AtALAD has high and a low molecular multimers ( Figures 3 - 4 , 6 ) we hypothesize that these multimers may reflect the active and inactive ALAD variants, as previously reported (Jaffe, 2020). However, we failed to verify these ALAD multimers by other experimental approaches, such size exclusion chromatography by Sepharose S250 and Superose 6 Increase 10/300 GL columns or by sucrose density ultracentrifugation. Thus, it remains an intriguing question how the AtALAD multimer is assembled and whether there is a control between an active and an inactive (or less active) form of the oligomeric complexes.

Apart from the first indication of oligomeric ALAD structure, we intended to verify the impact of redox control on ALAD activity and oligomerisation and began to identify the Cys residues, which are assigned to be responsible for the redox-dependent modified enzyme activity and for the formation of the quaternary structure of ALAD. It is hypothesized that thiol switches at certain Cys residues play a crucial role for redox-dependent modulation of enzyme activity and stability. To predict a potential role of the different Cys residues in the intra- and intermolecular disulfide bonding of ALAD, the recombinant ALAD mutants with either one or two Cys ➔ Ser substitutions were analyzed.

The redox studies with recombinant ALAD substitution mutants substantiate that the in vitro oligomerisation of ALAD under oxidizing conditions depends on existing Cys residues at different positions in the ALAD structure. The extent of oligomerisation varied in the untreated and oxidized in vitro samples in comparison to the predominant portion of monomers under reducing conditions ( Figure 6 ). In the SDS PAGE, the monomer of ALAD could be separated into differently migrating variants, which were distinguished between a reduced and three different oxidized forms ( Figure 6 ). The distinctive protein mobility of three oxidized forms in the denaturing gel is proposed by modified configurations of ALAD likely due to the single and combined occurrence of disulfide bonds.

Considering the positions of Cys residues in the A. thaliana ALAD model ( Figure 1 ) and their possibilities to form intramolecular disulfide bonds ( Supplementary Figure 2 ), Cys71 in the flexible N-terminal arm of ALAD is the best candidate for the intramolecular and intermolecular thiol switches and shows the strongest potential to form intramolecular disulfide bridges in vitro ( Figure 6 ). Based on the modified appearance of oxidized monomers of Cys-substituted mutants of ALAD, it can be proposed that Cys71 forms disulfide bonds with the two Cys residues at the positions 152 and 404. The assignment of the three differentially migrating ALAD bands under oxidizing conditions to either a fully oxidized ALAD variant or the mutant proteins containing one or two Cys substitution mutations is difficult and would not automatically correlate with the predicted possibility of a disulfide bond between two intramolecular Cys residues, as suggested by the structural model ( Supplementary Figure 2 ).

However, since ALAD(C251S) and ALAD(C404) and the double mutant lack up to two of the oxidized ALAD bands in the SDS gel, we suggest that the remaining oxidized ALAD protein contains still a possible disulfide bond between Cys71 and Cys152 and migrates as the ox1 form. The ALAD ox2-variant could be linked to a disulfide-independent redox-modification of Cys251. Based on the structural model of ALAD ( Figure 1 ) Cys251 can most likely not form an additional intramolecular disulfide bond with other Cys-thiol groups. Thus, Cys251 seems to be a potential site for another redox-dependent modification, such sulfenylation or an intermolecular disulfide bond. Redox-dependent proteomic studies revealed sulfenylated Arabidopsis ALAD (Akter et al., 2015). The ox3-variant could represent the combined disulfide bond formation between Cys71 and Cys152 or Cys404 and the redox modification on Cys251.

Incubation of ALAD under oxidizing conditions (CuCl₂, diamide) or reducing conditions (DTT) leads to the modified enzyme activities ( Figure 3 ; and Wittmann et al., 2018). The analysis of redox-dependent ALAD activity was advanced with the Cys-substitution mutants to correlate the finding on the redox-dependent oligomerisation of ALAD mutants with their redox-dependent catalytic activities. The oxidized forms of ALAD(WT) and C424S/C426S double mutant showed a 35% and 37% remaining activity of the reduced proteins, respectively. These two ALAD variants exhibited the strongest redox sensitivity of the enzyme activity. The activity assays exclude a putative role of the two 424/426 cysteines in redox-modulated activity. The ALAD(C424/C426) also showed a corresponding WT-like mobility in the non-reducing SDS-PAGE, excluding an impact on the oligomerisation of ALAD.

The loss of a thiol switch-dependent ALAD activity and modified structure become apparent for the mutant ALAD(C71S). This ALAD substitution mutant lost most of the redox sensitivity of ALAD activity, and their enzyme activity only slightly increased by 23% under reducing conditions. The loss of redox sensitivity can also be observed in the double mutants ALAD(C71S/C152S) and ALAD(C71S/C251S). However, the drastic reduction in enzyme activity of these two double mutants indicates the additional negative effects of each of the Cys substitutions on the overall activity of ALAD. The ALAD(C71S), ALAD(C152S) ALAD(C251S) show a 52%, 36% and 59% reduced activity, respectively, compared with ALAD(WT), while the double mutants exhibit a loss of 87% [ALAD(C71S/C152S)] and 83% [ALAD(C71S/C251S)] enzyme activity compared to ALAD(WT) ( Figure 5 ). If the dimerization of ALAD requires an intermolecular disulfide bond between the Cys residues, then a thiol switch is preferentially proposed at position 404. In the model of the dimer structure both residues have a distance of 14,5 Å ( Supplementary Figure 3 ). In the future, the question of how oxidizing conditions influence oligomerization, at least in in vitro conditions, and how oxidizing conditions lead to reduced activity should be resolved.

The plant ALAD sequences are characterized by the long N-terminal arm compared to the animal and bacterial homologs ( Figure 1 ) and mostly possess an AtCys71 homolog indicating the significance of this N-terminal Cys residue, while pea or soybean ALAD and AtALAD2 miss this equivalent Cys residue. A. thaliana mature ALAD possesses six Cys residues, while only four Cys were found in pea ALAD. The homologous Cys residues at position 71 and 424 of AtALAD are missing in the pea ALAD. It will be instructive to examine assembly and stabilization of the pea and Arabidopsis ALAD homologs to unravel the control mechanism of oligomerization and the switch between inactive and active ALAD. In contrast to AtALAD, pea ALAD seems not to form intramolecular disulfide bonds between Cys residues of the globular protein (Kervinen et al., 2000). As no dimer formation of the pea ALAD(C326A) mutant (homologous to Cys426 in AtALAD) was observed it was predicted that this Cys residue of ALAD potentially is involved in intermolecular disulfide bonding, although ALAD(C326A) showed complete oligomerization and WT like enzyme activity (Kervinen et al., 2000). These observations resembled the unmodified catalytic activity and formation of multimers of the ALAD(C424S/C426S) variant in comparison to WT.

These current findings raise several issues, which need to be addressed in future studies. What could be the role of a redox-sensitive Cys71 for ALAD inactivation and oligomerization? It should be kept in mind that oligomerization of ALAD is verifiable in native gels under reducing and oxidizing conditions ( Figures 4 , 6 ). It is evident that Mg²⁺ stabilizes the oligomers. The hindrance of oligomerization is only detectable when protein samples are subjected to denaturing gels. Thereby it became evident that ALAD(WT) requires reducing conditions to prevent oligomerization, while some ALAD Cys substitution mutants do not even assemble under oxidizing conditions.

Derived from our results, it is proposed that oxidizing conditions lead to intramolecular disulfide bonds formation in ALAD. In darkness, chloroplasts are characterized by oxidizing conditions and Mg²⁺ deficiency. These are preconditions for an adequate inactivation of ALAD. Given an oxidized environment in chloroplasts, inactive ALAD is achieved when assembly of ALAD monomers to octamers is attenuated. Then, AtALAD monomers could form an intramolecular disulfide bond and could assemble via dimers to the inactive hexamer. These predictions of a redox-dependent arrangement of ALAD subunits require further studies to verify the assembly and disassembly mechanism of AtALAD to build and stabilize the oligomeric ALAD structures. The structural arrangements between oligomers could be formed dependently or independently from redox-sensitive Cys residues, which are involved in intra- or intermolecular disulfide bridges.

In summary, the described Cys residues of ALAD seem to be not only relevant for the quaternary structure, but also for the modulation of the catalytic activity of ALAD during oxidized and reduced conditions. The presented data provide first indications for thiol-based redox regulation, which indicate a stimulatory effect on catalytic activity of ALAD under reducing conditions and the stabilization of oligomeric, inactive ALAD under oxidizing conditions. The formation and stability of the active ALAD octamer is supported by Mg²⁺. In future studies, the oligomerization and activity of Arabidopsis ALAD will be examined in planta by expression of the ALAD substitution mutants in the hemb1 mutant background.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

DW: Conceptualization, Investigation, Methodology, Writing – original draft. CW:. structual modelling, Writing -revised version, BG: Conceptualization, Funding acquisition, Supervision, Writing – original draft.