This enzyme can be found with different denomination: Di-Vinyl Reductase; DiVinylChlorophyllidea 8-Vinyl Reductase or 4VCR [4-Vinyl] Chlorophyllide a Reductase, but here in this work it will be just “DVR”. DVR converts divinylchlide a to monovinylchlide ain theChl biosynthetic route [9] [18] . The ethyl group at the C8 position (8E) of the macrocycle is produced by the reduction of a vinyl group (8V), catalyzed by an 8VR (8Vinyl reductase; EC 1.3.1.75), resulting in the production of an 8E pigment [17] . The DVR is divided into two types of unrelated enzymes (Figure 1), one in plant reductases, BciA belonging to the SDR family [24] , and also having a ligand-binding site using NADPH as electron donors. And the other enzyme, which is in cyanobacterial reductases: BciB which uses ferredoxin as an electron donors and is present in some organisms belonging to Chlorobia, cyanobacteria, eukaryotes and Proteobacteria. BciB has high homology with coenzyme F420-reducing hydrogenase only found in methanogenic archaea [25] - [27] . The BciB tree is one of the few cases where Chloroflexi and Chlorobia are not sister groups, with Chloroflexi, closer to Allochromatium vinosum and Chloracidobacterium thermophilum sequences. Also, BciB shows separation between the major proteobacterial clade and the other photosynthetic taxa [28] . The orthologous (BciA and BciB) genes form a well-supported clade that can be subdivided into two groups, one comprising GSB and Proteobacteria sequences and another with cyanobacteria and eukaryotic sequences. The only exception is the Acaryochloris marina sequence that clusters within the proteobacterial clade. Because of the broad distribution of NAD(P)H-binding sites (Rossmann folds “GxGxxG”) in proteins, database searching retrieved many additional hits, namely at the lower part of the tree [28] , but in the protein alignment here for the phylogenetic trees, the DVR, and the other nitrogenase-like enzymes, showed no significant presence of Rossmann folds, except in the case of the L subunit (DPOR).

In the Chl biosynthesis, the final reaction that differentiates the angiosperms from the rest of the photosynthetic organisms [3] forms the chlorine structure of Chla at the Pchlide. This step isa key regulatory step in the Chl biosynthetic pathway in plants and bacteria, which has been extensively studied [21] [29] - [33] . This reaction is performed by the enzyme: Light-Dependent Protochlorophyllide (Pchlide) Oxidoreductase (LPOR: EC 1.3.1.33), also named NADPH-Pchlide Oxidoreductase, which catalyzes the light-dependent trans addition of a hydrogen from NADPH to the carbon atom carrying the propionic side across the stereo-specific reduction of the C₁₇ = C₁₈ double bond of the ring D (Figure 1) of Pchlide to produce Chlide [21] [29] [33] . This reaction, requires both light (absorption maximum 638 - 650 nm) and NADPH for activity [3] [21] [34] . The LPOR polypeptide binds both NADPH and Pchlide, but the reduction does not occur until the protochlorophyllide molecule absorbs light at 628 - 630 nm [35] . LPOR is composed of a single polypeptide that belongs to Short-chain Dehydrogenase/ Reductase (SDR) family [30] [33] . LPORis the enzyme responsible for the greening of angiosperms and is the only Pchlide reductase to operate in angiosperms [21] [31] [32] [36] . This starts to act when triggered by light, and plays an important regulatory role in angiosperm development, since it functionally acts as a gate in the biosynthetic pathway allowing Chl synthesis only when the plant is illuminated [37] . According to Yang & Cheng [38] the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms, and the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer proving the finding of Suzuki & Bauer [37] , which defended that LPOR evolved before the advent of eukaryotic photosynthesis and that LPOR did not arise to fulfill a function necessitated either by the endosymbiotic evolution of the chloroplast or by multicellularity; rather, it evolved to fulfill a fundamentally cell-autonomous role. Based on the protein sequence alignments, Wilks and Timko [30] identified two conserved residues (Tyr-275 and Lys-279) within the active site of the enzyme and also showed that they are critical foractivity of LPOR.

Dark-operative (or Light-Independent) Pchlide Oxidoreductase (DPOR: EC 1.3.7.7) is the other enzyme that is involved in the reduction of the penultimate step of Bchl/Chlbiosynthesis, [39] , DPOR catalyzes the same stereo-specific reaction of C₁₇ = C₁₈ double bound of Pchlide ring D (Figure 1) to form Chlidea absence of light [31] in oxygenic photosynthetic organisms [40] . Genetic and sequencing studies have showed that DPORwas first studied in purple bacteria [10] [41] , where it was recognized to be essential for synthesis of Bchl in the dark. DPOR is a nitrogenase-like enzyme with three subunits B, L and N [32] [35] [40] - [45] , that code for putative subunits of the DPOR [11] [42] , which are encoded by Bchl genes (BchB, BchL, and BchN) in photosynthetic bacteria genomes or by the ortholog Chlgenes (ChlB, ChlL, and ChlN) in the plastid genomes of photosynthetic eukaryotes. These subunits are divided into two protein complexes, theL-protein (BchL/ChlL homodimer), a reductase component and an ATP dependent reductase specific for other catalytic component.The other complex is denominated NB-proteincomplex (a BchN-BchB/ChlN-ChlB heterotetramer), which provides the catalytic centers for the double-bond reduction of the Pchlide D-ring [23] [43] [44] [46] . It has already been demonstrated that ChlN and ChlB proteins are soluble and provide the catalytic site for the Pchlide reduction, and each NB-protein provides the catalytic sites for Pchlide reduction and carries a pair of 4Fe-4S clusters that would mediate between the cluster of L-protein and Pchlide [47] . This cluster called “NB-cluster” is unique because it is coordinated by three Cysteine (Cys) residues from BchN (BchN-Cys₂₆, BchN-Cys₅₁, BchN-Cys₁₁₂) and one Aspartic Acid (Asp) residue from BchB (BchB-Asp₃₆). DPORis distributed widely in the photosynthetic organisms since primitive anoxygenic photosynthetic bacteria as the sole photosynthetic enzyme [48] . It is also found in cyanobacteria, algae, mosses, liverworts, and gymnosperms. In each of these organisims DPOR has the company of its homologous LPOR to reduce the C₁₇ = C₁₈ double bound [3] [32] [46] . DPORis considered a unique iron-sulphur enzyme that forms substrate radicals followed by sequential proton and electron-transfer steps [33] . The catalytic mechanism of DPOR includes the electron transfer from a “plant-type” [2Fe-2S] ferredoxin onto the dimeric DPOR subunit, BchL, carrying an intersubunit [4Fe-4S] redox center coordinated by Cys₉₇ and Cys₁₃₁ in Chlorobaculum tepidum. Wätzlich et al. [49] , showed that in DPOR were found Lys₁₀ in the phosphate-binding loop (P-loop) and Leu₁₂₆ in the switch II region, what is essential for DPOR catalysis and analogous to nitrogenase. Electrons from the 4Fe-4S cluster of BchNB/ChlNB are transferred directly onto the Pchlide substrate at the active site of DPOR [49] . Evolutionary studies say that DPOR has been disappearing in the evolution pathway of plants, from gymnosperms to angiosperms [31] [32] [50] - [55] . According to [56] one of the reasons may be the low temperature, this has not been confirmed experimentally so far, and the fact that light-dependent pathways reduce Pchlide which may confer a selective advantage in certain environments for angiosperms. Some authors [43] [57] [58] suggest that the ChlL protein is associated with membranes and that it may function as an adenosine triphosphate-dependent electron donor, and Reinbothe et al. [22] suggests the L-protein of DPOR might function to sense the partial oxygen pressure in response to the light environment, thus providing a molecular tool to switch from oxygen-sensitive to oxygen-insensitive Pchlide reduction, confirming again this oxygen sensibility of the DPOR, specifically the L-protein subunit. NB-protein is stable in an aerobic condition [47] . Crystal structures of the DPOR components show that the shortest distance between an NB-cluster and Pchlide is 10.0 Å, which is close enough for permitting the through-space electron transfer reaction [59] . A primary, single electron transfer from the 4Fe-4S cluster of L-protein to NB-cluster of NB-protein occurs and it is similar to that from the 4Fe-4S cluster of Fe-protein to the P-cluster of MoFe-protein in the nitrogenase complex [43] [60] . The activity of DPOR is dependent upon both ATP hydrolysis and a reductant, which is most likely ferredoxin in vivo, and also dependent on the reductant dithionite [43] [44] . Crystallographic analysis of L-protein and NB-protein complex from Prochlorococcus marinus suggested that a water molecule just above C₁₈ is the direct proton donor for C₁₈ rather than the C₁₇-propionate in the Prochlorococcus NB-protein [61] , however contribution of the water molecule has not yet been experimentally proven to be critical for Pchlide reduction in the Prochlorococcus DPOR. Results obtained so far in Rodobacter capsulatus DPOR support the reaction mechanism of the C₁₇-propionate as the proton donor to C₁₈ [33] .

In most photosynthetic organisms, the chlorin ring structure of Chla is formed by the reduction of the porphyrin D-ring by the DPOR. Subsequently, the chlorine ring B (Figure 1) is reduced in Bchl biosynthesis to form 3-vinyl bacteriochlorophyllide which has a bacteriochlorin ring structure [23] . This reduction is made by the enzyme: Chlide Reductase (COR: EC 1.3.99.35) which has three subunits: bchX, bchY and bchZ [53] , and reduces the C₇ = C₈ double bond of Chlide [62] . According to Nomata et al. [23] structural changes in COR have special effects on the spectral properties of these compounds enabling them to absorb infrared light to perform anoxygenic photosynthesis.COR from the bacterium Rhodobacter capsulatus is described to be able to also reduce the 8V group of Chlidethus considered the third class of 8VR (DVR), referred as a COR-type reductase [62] . R. capsulatusalso contains an orthologue of BciA (the translated sequence of which is 61% identical and 72% similar to BciA from R. sphaeroides). It is likely that organisms with 8E-BChls use COR to reduce the 8V group of any Chlide molecule that has bypassed the conventional 8VR. This mechanism may also account for the lack of any BciA or BciB orthologues in the genomes of 8E BChla-producing Roseiflexus species [63] of green non-sulfur bacteria.

The DPOR and the COR enzymes show the most similarities with each other (Table 1), both convert porphyrin (Pchlide) to bacteriochlorin (3-vinyl Bchlide a) in Bchl a biosynthesis [47] and both are composed of three subunits [40] . In the amino acid sequence alignments of BchX proteins and the closely related Bchlor ChlL subunits of DPOR, both cysteinyl ligands responsible for 4Fe-4S cluster formation and residues for ATP binding are conserved [65] . Wätzlich et al. [49] presents, that COR subunit BchX forms a redox-active inter subunit cluster analogous to that described for DPOR subunits of Bchlor ChlL. In addition, the Tyrosine (Tyr₁₂₇) is proved to be essential for DPOR catalysis, where this surface-exposed residue is directly involved in protein-protein interaction and is responsible for the inter subunit electron transfer [49] . Gene duplication of an ancient reductase gave rise to a nitrogenase (NifH) and a Bchl/Chl branch. This ancient reductase (Chl/Bchl) of the Bchl/Chl path has thus evolved into the current BchX₂ and BchL₂/ChlL proteins. This evolutionary process came along with the appearance of subunits BchNB/ChlNB and BchYZ, responsible for the specific reduction of rings B and D, respectively [49] . BchX and BchL share 34% amino acid sequence identity [53] , also proved by the phylogenetic tree (Figure 2) where the two subunits are close to each other. Relatively to COR, which is also very similar related to DVR (Table 1), is also what was expected after know that it is able to reduce the 8 V group of Chlide, because of that is being considered the third class of DVR [62] . Anaerobic conditions are required for the maximum activity of DPOR to complement the loss of LPOR [66] . Analysis of DPOR and LPOR enzymatic activity, in a variety of species, has demonstrated several functional differences in these enzymes, showing that coordination and regulation of DPOR and LPOR activity are responsive to environmental conditions, which allow the

photosynthetic organisms to maintain a high level of Chl biosynthesis under distinct and changing conditions in their environment [67] . The reductions made by LPOR, and BciA (DVR) enzymes occur in the former process with NADPH as a hydride-donating cofactor (Table 1), while DPOR, BciB (DVR) and COR enzymes catalyze the reduction through the latter process with ferrodoxin enzymatically reduced by NADPH as an electron donor [68] . After analyze at all the characteristics mentioned before, the comparison between these four enzymes shows that the LPOR is the one that has the less similarities related to the rest of this group (Table 1). LPOR is the most distinct according to the multiple sequence alignment with the other enzymes (DPOR, COR and DVR) which are closely related when comparing with each other. They have several characteristics in common (Table 1).

Nitrogenase (dinitrogenase) is a complex enzyme responsible for biological nitrogen fixation which converts the atmospheric dinitrogen (N₂) into ammonia (NH₃) [69] - [71] , in N₂-fixing organisms (diazotrophs). A typical nitrogenase is an α₂β₂ tetramer encoded by NifH, NifD and NifK and containing an iron-molybdenum cofactor, FeMoCo [72] [73] . Nitrogenase is an enzyme that is divided into two protein complexes, oneisFe-protein, which is an ATP-dependent reductase specific for the MoFe-protein and also carries one 4Fe-4S cluster bridged between these two complexes. The other complex is theMoFe-protein, which provides the catalytic centers. It has two types of metalloclusters: the P-cluster (an [8Fe-7S] cluster) and the FeMo-cofactor comprising 1Mo-7Fe- 9S-X homocitrate. The Fe-protein is reduced by ferredoxin or flavodoxin and then the electrons are transferred from the [4Fe-4S] cluster of the Fe-protein to the FeMo-cofactor of the MoFe-protein via the P-cluster. The enzymes DPOR and COR (Table 1) are considered twonitrogenase-like enzymes because they share significant amino acid sequence homology to the corresponding subunits of nitrogenase. Whereas subunits BchL/ChlL, BchX and NifH exhibit a sequence identity at the amino acid level of 33%, subunits BchN/ChlN, BchY, NifD, and BchB/ChlB, BchZ, and NifK, respectively, show lower sequence identities of 15%. The DPOR’s NB-protein shows more similarity with the nitrogenase homologous MoFe protein (a NifD-NifK heterotetramer) of nitrogenase [23] [31] [40] [43] [44] [46] [47] [53] [74] , contrarly, the L-protein (DPOR) is most structurally related to Fe-protein (a NifH homodimer) (33% identity and 50% similarity) [44] . Other authors [23] [33] [40] [43] [44] [60] discovered more cases of significant similarities between them such as all of them are dependent on the presence of ATP and reductant dithionite. Nomata et al. [23] showed that the in vitro assay catalytic mechanism of COR strongly resembles DPOR catalysis.X-ray crystallographic, electron paramagnetic resonance, mutagenesis studies and sequencing analysis showed more indepth information about the structure of some of theseenzymes, confirming some mentioned above and new ones. For example the N and B proteins (DPOR) have shown that they do not have the requisite number of conserved Cys to assemble a P-cluster or the FeMo cofactor of the nitrogenase NifK and NifD heterotetramer. Instead, the conserved Cys arrangement in ChlN/BchN and ChlB/BchB seems to be more consistent with a cluster similar to the one found in the nitrogenase accessory proteins NifE and NifN [44] . TheseCys residuesligate the intra subunit 4Fe-4S cluster, that is chelated by the two protomers of L-protein [40] . These Cysresidues are also conserved in ChlL/BchLwhat also happen to the nitrogenase NifH dimer [44] [45] . In all the nitrogenase iron protein’s sequences and in bchX, the position 100 is occupied by an Arginine (R) residue and in both BChland ChlL it is Tyrosine (Y) [13] . The Tyr₁₂₇ is found conserved in all Bchl and Chl proteins, whereas the nitrogenase system, as well as the COR system makes use of an Arginine at the identical position [49] . These similarities happens because DPOR has evolved from ancestral genes common to nitrogenase and is distributed among anoxygenic photosynthetic bacteria, cyanobacteria, Chlorophytes, Pteridophytes, Bryophytes, and Gymnosperms, which until now has made them carry a lot of similarities [13] [75] . Another feature that Nitrogenase, COR and DPOR have is the oxygen sensitivity (Table 1), which can be irreversibly destructed by oxygen [40] . Through the evolutionary tree of the organisms, besides the fact of some domains being kept highly conserved [44] [45] [49] , the DPORhave got smooth changes with the time, which in future can decrease significantly its oxygen sensibility, but still, the electrons from the [4Fe-4S] cluster of BchNB/ChlNB, that are transferred directly onto the Pchlide substrate at the active site of DPOR [49] , a reaction that is potentially dangerous under oxygen-rich conditions because of the substrate radical that would be a source of reactive oxygen species that would cause severe damage to cells [40] . The rapid rise in global oxygen level may have also driven the evolution of heterocysts to protect nitrogenase from oxygen, that when reaches levels over 0.3% (v/v), the “Pasteur point”, considered the level above which the activity of DPOR is functionally insufficient, and the LPOR becomes essential to survive, in the organisms that have both DPOR and LPOR [66] [76] [77] . Some species, as cyanobacteria, to cope with environmental generated reactive oxygen specieshave developed effective protection mechanisms such as catalases, peroxidases, superoxide dismutases, A-type flavor proteins (Flv1 and Flv3) [66] An example, in Cynechocystis sp. PCC6803 which are essential for photoreduction of oxygen to water and are probable candidates for the DPOR protection machinery and these mechanisms are believed to protect not only L-protein but also NB-protein from oxygen, upon exposure to air, where the gradual decrease in activity of purified NB-protein is much slower than that of L-protein [77] . DPOR seems to have become dependent on the protection mechanism rather than evolved to acquire oxygen tolerance, so, the presence of such mechanisms to protect DPOR could contribute to the evolutionary persistence of DPOR in oxygenic photosynthetic organisms. DPOR no longer operates in conditions where oxygenic photosynthesis is very active and cellular oxygen levels are very high [66] .