The C-Terminal Extension of Ferrochelatase Is Critical for Enzyme Activity and for Functioning of the Tetrapyrrole Pathway in Synechocystis Strain PCC 6803

Abstract

Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the ⌬H324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the ⌬H324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the ⌬H324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the ⌬H324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the "extra" flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.

Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes

“…The better viability of the gun4 Ϫ cells under low light or microaerobic conditions may be explained by the increased accumulation of P IX in this mutant (Table 1) as P IX is known to cause photooxidative damage (27). Recently, a very similar phenotype has been observed in a Synechocystis 6803 mutant that also accumulated large quantities of P IX , resulting from a mutation in the ferrochelatase gene (28). However, at low light intensities under microaerobic conditions no further improvement in Chl accumulation was observed, with the gun4 mutant accumulating about 25% of the wild-type Chl content.…”

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…The sensitivity of Gun4-less mutants to light and changes in ALA synthesizing capacity led to a hypothesis that Gun4 binds excessive porphyrins, which serves as a signal to down-regulate ALA formation (19,20). Although this model can explain reduced ALA synthesis in Gun4 or MgCh mutants (40), it is not consistent with the massive accumulation of P IX in ferrochelatase mutants (8,41).…”

Functional Analysis of Light-harvesting-like Protein 3 (LIL3) and Its Light-harvesting Chlorophyll-binding Motif in Arabidopsis

“…Although these data do not exclude the possibility that the amino acid modification exerted conformational changes on other parts of LIL3, we think this is unlikely because the experiments with LIL3(TM APX ) indicated that the conformation of the TM domain and that of the other part of LIL3 were independent from each other. In the case of cyanobacterial FC, removal of the LHC motif from the FC sequence resulted in the dissociation of the FC dimer into monomers (23). The results led us to hypothesize that the modification of the LHC motif in TM LIL3 also affects the oligomerization of LIL3-GGR complexes.…”

“…At least three specific functions have been postulated for the LHC motif: membrane anchoring, providing binding sites of tetrapyrroles, and facilitating protein-protein interactions (5,22). The function of the LHC motif of cyanobacterial FC has been studied at both the in vivo and in vitro levels (22)(23)(24). Although removal of the LHC motif from the FC sequence did not affect its catalytic activity, it resulted in a dissociation of the dimeric FC into monomers (22) and in the increased turnover of the FC reaction (24).…”