Photoprotective role of NADPH:protochlorophyllide oxidoreductase A

Abstract

A homology model of NADPH:protochlorophyllide (Pchlide) oxidoreductase A (POR; E.C. 1.3.33.1) of barley is developed and verified by site-directed mutagenesis. PORA is considered a globular protein consisting of nine α-helices and seven β-strands. The model predicts the presence of two functionally distinctive Pchlide binding sites where the pigment is coordinated by cystein residues. The pigment bound to the first, high-affinity Pchlide binding site is used for the formation of the photoactive state of the enzyme. The pigment bound to the second, low-affinity Pchlide binding site is involved in the PORA:PORB interaction, allowing for resonance energy transfer between the neighboring PORs in the complex. In the in vitro reconstituted light-harvesting POR:Pchlide complex (LHPP), light absorbed by PORA-bound Pchlide b is transferred to PORB-bound Pchlide a. That induces the conversion of Pchlide a to chlorophyllide (Chlide) a. This energy transfer eliminates the possibility of Pchlide b photoreduction and prevents that excited triplet states of either Pchlides a or b accumulate and provoke singlet oxygen production. Together, our results provide a photoprotective role of PORA during greening.

A key enzyme in the light-induced greening of higher plants is NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR). In angiosperms such as barley, Arabidopsis (1, 2), tobacco (3) and Amaranthus tricolor (4), and gymnosperms such as pine species (5–7), several por gene families were identified that encode highly conserved POR polypeptides. PORA represents the negatively light-regulated POR enzyme whose level drops as a result of the concerted effect of light at the levels of transcription, mRNA stability, plastid import, and protein degradation after light-induced catalysis (8–11). PORB, the second POR protein identified in barley and Arabidopsis (1, 12), is constitutively expressed in dark-grown, illuminated, and light-adapted plants (see ref. 11 for review). A third light-induced por gene was discovered in Arabidopsis thaliana that complements PORB in green plants (13, 14).

PORA and PORB constitute larger light-harvesting POR:Pchlide complexes dubbed LHPP in the prolamellar body of etioplasts (15–18). These complexes additionally interact with galacto- and sulfolipids and establish two spectral forms of Pchlide: Pchlide-650/657 and Pchlide-628/632 (the first number indicates the absorption maximum, the second shows the respective fluorescence emission maximum). Pchlide-650/657 is also called photoactive Pchlide because it can be converted to Chlide-684/690 with a single, 1-ms flash of white light. Pchlide-628/632, by contrast, is nonphotoconvertible and named photoinactive Pchlide. Energy transfer takes place from photoinactive Pchlide to photoactive Pchlide and from photoinactive Pchlide to Chlide (summarized in ref. 19). Similar spectral pigment species and interconversions have been described for reconstituted PORA:PORB-lipid supracomplexes (15–18). All information available at the moment suggests that the native and reconstituted complexes are structurally and functionally identical. The two major roles attributed to LHPP are light trapping and the dissipation of excess light, both of which are needed when dark-grown seedlings de-etiolate (15). In the present work, we provide direct evidence for a photoprotective role of PORA during greening.

Results

Homology Modeling of Barley PORA and PORB.

The barley PORA and PORB are related in their primary amino acid sequences to short-chain dehydrogenases (SCDs) (refs. 20–22 and see Fig. 1A for sequence comparison). For these enzymes, x-ray structures have been obtained (23–25), allowing homology models to be constructed with MODELLER 9v3 software and 7-α-hydroxysteroid dehydrogenase (AHI) from Escherichia coli as a template.

Fig. 1.

Homology models of the barley PORA and PORB. (A) Primary amino acid sequences of the PORA and PORB of barley and POR of Synechocystis and their relationship to AHI of E. coli. α-Helices (marked α-A–I) and β-strands (marked β-1–7) are indicated. The GxxxGxG and YxxxK motifs implicated in NADPH binding and the conserved Cys residues (referred to as Cys-1, Cys-2, Cys-3, and Cys-4) are highlighted. Numbering refers to the full-length PORA and PORB polypeptides containing their NH₂-terminal transit peptides for plastid import (12) and synPOR, which does not contain a comparable NH₂-terminal extension (22). Identical amino acids are given in white letters on red background, whereas conserved residues are highlighted with red letters in black frames. Dots indicate gaps introduced during sequence alignment. Note the presence of the so-called extra loop, establishing an oligomerization domain of the PORA (amino acids 217-252) and PORB (amino acids 225-260) (ref. 18), and its absence from AHI of E. coli. (B and C) Structural models of the barley PORA (B) and PORB (C). α-Helices and β-strands are shown in red and green in the ribbon representation, respectively. Positions of the YxxxK and GASSGLG motifs and the conserved Cys residues (Cys-1–4) are indicated. The extra loop was conceptually incorporated as helix-turn strand. The models depict the nonsubstrate- and noncosubstrate-complexed states of the PORA and PORB.

The obtained PORA and PORB structural models are shown in Fig. 1 B and C. Accordingly, the PORA and PORB of barley are predicted to be globular proteins consisting of nine α-helices and seven β-strands. Similar α-helices and β-strands are present in POR of Synechocystis (synPOR) and are marked α-(A-I) and β-(1–7), respectively, in Fig. 1. Amino acid conservation in synPOR and the barley PORA and PORB is almost perfect in the so-called Rossmann fold representing the adenine binding cleft in the SCDs. Amino acid residues already established as being necessary for structural and functional reasons in the SCDs are also conserved in synPOR and the barley PORA and PORB (Fig. 1). It is assumed that the highly conserved Tyr and Lys residues in the YxxxK motif, in conjunction with the GASSGK/LG motif, establish the enzyme catalytic pocket and participate in the proper coordination of NADPH. The ε-amino group of the Lys side chain hereby is proposed to interact with the hydroxyl group of Tyr and to lower its pK_a such that a proton can be donated during the catalytic mechanism. In addition, both amino acid residues are implicated in the formation of the POR photoactive state with Pchlide (22, 26–28).

The participation of one or two Cys residues in Pchlide binding has been proposed (22, 29). SynPOR contains six Cys residues of which Cys-33, Cys-85, Cys-195, and Cys-222, henceforth referred to as Cys-1, Cys-2, Cys-3, and Cys-4, respectively, have counterparts in the PORA and PORB of barley (Fig. 1A and Table S1).

Evidence for the involvement of Cys residues in pigment binding and catalysis has come from chemical modification of Cys residues with N-phenylmaleimide and other sulfhydryl group-modifying compounds (29). For example, upon treatment of synPOR with these agents, a decrease in the enzyme activity was observed (22). In the proposed structural models (Fig. 1 B and C), at first glance all four Cys residues in the PORA and PORB are in close proximity to the active site and thus could be involved in Pchlide binding. Cys-1 is facing the GASSGK/LG motif, whereas Cys-3 is juxta-positioned to the YxxxK motif participating in the catalytic mechanism. Cys-2 has no counterpart in POR of pea and was therefore disregarded as Pchlide binding site, whereas Cys-4 is present between β-strand β6 and helix α-G and just opposite to the extra loop, establishing an oligomerization domain between the PORA and PORB (18).

Bacterial Expression, Purification, and Characterization of Barley PORA and PORB.

To identify which Cys residue in the PORA could provide a Pchlide binding site, we chose an in vitro mutagenesis approach. DNAs encoding (CysX→Ala)-PORA (X = 1, 2, 3, or 4) proteins were generated as described in Materials and Methods, sequenced, and subcloned into the pQE30 vector. Cloning was made in a way such that the resulting proteins would no longer contain their transit peptides for plastid import but to bear hexa-histidine [(His)₆] tags at their NH₂ termini, allowing for their ease of purification. After transformation into E. coli strain XL1-Blue, PORA and PORB protein expression was induced with isopropyl β-D-thiogalactoside. After 3 h, bacteria were harvested by centrifugation and lysed, and the soluble fraction containing PORA and PORB was obtained by differential centrifugation. After a step of Ni-NTA agarose chromatography, the final protein fractions were ≈95% pure and contained only minor contaminants of bacterial proteins [supporting information (SI) Fig. S1]. Pilot experiments (Table S2 and Fig. S2) reveal that thus expressed and purified WT PORA and PORB display the same stringent substrate specificities for Pchlide a and b as the wheat germ-translated proteins used previously (15, 17). Each POR protein bound two Pchlide molecules per enzyme monomer of which only one was photoconvertible (Table S2 and Fig. S2).

Substrate Binding to PORA Mutant Proteins.

Bacterially expressed and purified (Cys-1→Ala)-PORA, (Cys-2→Ala)-PORA, (Cys-3→Ala)-PORA, and (Cys-4→Ala)-PORA were incubated with Pchlide b and NADPH in the dark for 15 min. Free and weakly bound pigments were removed by gel filtration on Sephadex G15 equilibrated with assay buffer lacking sucrose. Sucrose has a remarkable effect on the binding of pigment to the PORA and PORB and stabilizes weakly bound pigments (Table S2 and ref. 30). (CysX→Ala)-PORA-pigment-NADPH ternary complexes eluted with the flow-through in turn were extracted with acetone. The pigments were identified and quantified by HPLC, room temperature absorption, and fluorescence spectroscopy (17).

Table 1 shows that, similar to the WT PORA (Table S2), the (Cys-1→Ala)-PORA and (Cys-2→Ala)-PORA each bound two Pchlide b molecules per enzyme monomer, only one of which was photoconvertible and transformed to Chlide b (see Fig. S3 A and B). Meanwhile, (Cys-3→Ala)-PORA and (Cys-4→Ala)-PORA were able to bind only one Pchlide b molecule per enzyme monomer (Table 1 and Fig. S3 C and D). While Pchlide b attached to (Cys-3→Ala)-PORA was nonphotoconvertible and only weakly bound to the enzyme, Pchlide b was tightly bound to (Cys-4→Ala)-PORA and readily converted to Chlide b upon illumination (Table 1 and Fig. S3 C and D).

Table 1.

Pigment binding and conversion characteristics of (CysX→Ala)-PORA modified proteins

Protein	Pchlide b, pmol per μg POR protein
	+ Sucrose		− Sucrose
	Dark	Flash	Dark	Flash
(Cys1→Ala)-PORA	54.40 ± 0.20	27.20 ± 0.04	27.20 ± 0.04	n.d.
(Cys2→Ala)-PORA	54.20 ± 0.15	27.30 ± 0.16	27.20 ± 0.04	n.d.
(Cys3→Ala)-PORA	27.20 ± 0.04	27.30 ± 0.18	n.d.	n.d.
(Cys4→Ala)-PORA	27.25 ± 0.06	n.d.	27.40 ± 0.24	n.d.

One microgram of PORA corresponds to ≈27.72 pmol of enzyme. ³⁵S-(CysX→Ala)-PORA-pigment-NADPH complexes were reconstituted in the presence or absence of 350 mM sucrose and subjected to gel filtration on Sephadex G15. POR-pigment-NADPH complexes eluted with the flow-through were then either kept in the dark or exposed to a saturating, 1-ms flash of white light. After extraction with acetone, pigments were identified and quantified by fluorescence spectroscopy. Standard deviations were calculated from five independent experiments. n.d., not detectable Pchlide b levels.

Assembly of PORA Mutant Proteins into Larger Complexes.

Reconstituted (CysX→Ala)-PORA-Pchlide b-NADPH complexes were mixed with equimolar amounts of WT PORB-Pchlide a-NADPH ternary complexes. After incubation for 15 min in the dark, the mixtures were size-fractionated on a Superose 6 column equilibrated with assay buffer containing sucrose that was used to stabilize weakly bound pigments (30). As a control, ternary complexes containing the WT PORA and PORB were used.

Fig. 2 shows results obtained for PORB and the WT PORA and combinations of PORB and the different (CysX→Ala)-PORA derivatives. Fig. 2A depicts complex formation for the WT PORA and PORB (fractions 3–5). Free, nonassembled PORA- and PORB-pigment ternary complexes were eluted in fractions 17 and 18, respectively. When the assembly was carried out with the (Cys-1→Ala)-PORA, (Cys-2→Ala)-PORA, and (Cys-3→Ala)-PORA, the results were similar to those obtained for the WT PORA. All three modified proteins formed larger complexes that were similar in size to those obtained for WT PORA (Fig. 2, compare B a–c with A). Protein quantification demonstrated similar ≈5:1 stoichiometries of PORA/PORB in the reconstituted supracomplexes (Fig. 2C). On the other hand, (Cys-4→Ala)-PORA produced drastically less PORA:PORB supracomplexes that, in most experiments, were at the limit of detection (Fig. 2Bd).

Fig. 2.

Assembly of (CysX→Ala)-PORA proteins with PORB. Reconstituted PORA-Pchlide b-NADPH, (CysX→Ala)-PORA-Pchlide b-NADPH subcomplexes were incubated with equal amounts of PORB-Pchlide a-NADPH ternary complexes for 15 min in the dark. Then the assays were subjected to gel filtration on Superose 6. Individual fractions were harvested and aliquots taken for SDS/PAGE and Western blotting using an ECL system. (A) Detection of established higher molecular mass PORA:PORB complexes containing WT PORA and PORB (fractions 3–5). Fractions 17 and 18 show unassembled PORA-pigment-NADPH and PORB-pigment-NADPH complexes. (B) Detection of higher molecular mass PORA:PORB complexes containing PORB and the (Cys-1→Ala)-PORA (a), (Cys-2→Ala)-PORA (b), (Cys-3→Ala)-PORA (c), and (Cys-4→Ala)-PORA (d) proteins. (C) Quantification of (CysX→Ala)-PORA (numbered 1–4 for the corresponding Cys residues) and PORB in the established higher molecular mass complexes. Percentages refer to the total amount of signal obtained on the films, including assembled and nonassembled POR–pigment complexes. The ratio indicates the relative amounts of the (CysX→Ala)-PORA to PORB in the recovered higher molecular mass complexes.

Pigment Conversion in PORA:PORB Supracomplexes.

Oligomeric PORA–PORB protein complexes reconstituted as described above were supplemented with a lipid mixture containing monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol (58:36:6 mol%), which had been prepared from pigment-depleted prolamellar bodies of barley etioplasts (31). Then the samples were analyzed further in two ways. To monitor the formation of lipid-containing higher molecular mass complexes, one aliquot was subjected to nondenaturing, analytical PAGE (NA-PAGE) (17). The other aliquots were subjected to fluorescence emission spectroscopy at 77 K at the excitation wavelength of 440 nm, which was used to trace the presence of Pchlide-650/657 (32). For (Cys-4→Ala)-PORA, which did not assemble into larger complexes with PORB (see Fig. 2), pigment- and NADPH-complexed PORA and PORB ternary complexes were directly added at a 5:1 stoichiometry to the lipid mixture and analyzed identically.

Fig. 3 depicts low-temperature fluorescence emission spectra of PORA:PORB supracomplexes that had been reconstituted with PORB and the different (CysX→Ala)-PORA proteins. Apparently, (Cys-1→Ala)-PORA and (Cys-2→Ala)-PORA were capable of reconstituting Pchlide-650/657 (Fig. 3 A and B, solid lines). In either case, the established Pchlide-650/657 was photoactive and converted into Chlide-684/690 upon flashing the samples. The level of Pchlide-628/632 remained unchanged (Fig. 3 A and B, dotted lines).

Fig. 3.

Low-temperature fluorescence analysis of reconstituted, lipid-containing PORA:PORB supracomplexes before and after flash light illumination. PORA:PORB supracomplexes containing the different pigment-complexed (CysX→Ala)-PORA proteins were reconstituted as described in Fig. 2 and supplemented with a mixed galacto- and sulfolipid fraction isolated from pigment-depleted prolamellar bodies of barley etioplasts. Then the samples were cooled to 77 K and analyzed by fluorescence emission spectroscopy at an excitation wavelength of 440 nm. The curves show spectra obtained for (Cys-1→Ala)-PORA-containing assays (A), (Cys-2→Ala)-PORA-containing assays (B), (Cys-3→Ala)-PORA-containing assays (C), and (Cys-4→Ala)- PORA (D)-containing assays before (solid lines) and after (dotted lines) a saturating 1-ms flash of white light.

For (Cys-3→Ala)-PORA and (Cys-4→Ala)-PORA, a different result was obtained. The (Cys-3→Ala)-PORA-containing complex produced a broad fluorescence peak emitting at ≈635 nm (Fig. 3C, solid line). A more precise spectroscopic analysis based on the second derivative and Gaussian deconvolution resolved the 635-nm band to consist of four Pchlide forms, with fluorescence emission maxima at 628, 632, 635, and 642 nm, designated Pchlide-F628, Pchlide-F632, Pchlide-F635, and Pchlide-F642, respectively. Upon flash light illumination, almost no visible change occurred in the low-temperature Pchlide fluorescence emission spectrum, except for a minor decrease in the Pchlide-F635 and Pchlide-F642 peaks. In addition, a new pigment fluorescence peak appeared with an emission maximum at 672 nm (Fig. 3C, dotted line). Pigment analysis in acetone at room temperature by fluorescence spectroscopy and HPLC on C30 RP columns showed that small amounts of Chlide a had been produced (data not shown). By contrast, no Chlide a was detectable for assays containing (Cys-4→Ala)-PORA (Fig. 3D and data not shown).

Light-Induced Dissociation of PORA:PORB Supracomplexes.

The aliquots of the reconstituted PORA:PORB:lipid complexes that were subjected to nondenaturing, analytical PAGE were detected by the blue light-induced pigment fluorescence and Western blotting using a POR antiserum (33), respectively. Fig. 4 A and C shows that (Cys-1→Ala)-PORA, (Cys-2→Ala)-PORA, and (Cys-3→Ala)-PORA all were present in terms of larger complexes exhibiting strong pigment fluorescence. By contrast, no fluorescing supracomplexes were found for (Cys-4→Ala)-PORA (Fig. 4 A and C) that had previously been demonstrated to be assembly-incompetent. Nevertheless, (Cys-4→Ala)-PORA was present as a pigmented complex, as evidenced by the appearance of a corresponding fluorescence band under excitation with blue light (Fig. 4C). Also, the fluorescence of PORB indicated the presence of pigment. SDS/PAGE of aliquots of the incubation mixtures proved that similar amounts of (Cys→Ala)-PORA and PORB proteins were present (Fig. 4E).

Fig. 4.

Nondenaturing, analytical PAGE of lipid-containing PORA:PORB supracomplexes. Pigment-complexed PORB and (CysX→Ala)-PORA proteins were reconstituted into their lipid-bound states as described in Fig. 3. Then aliquots of the assays were either immediately loaded onto a nondenaturing, analytical polyacrylamide gel or flashed before electrophoresis. Aliquots of the different samples were inspected under blue light for their pigment fluorescence and subsequently subjected to Western blotting. (A and B) Western blot of POR-related proteins in assays containing PORB and (Cys-1→Ala)-PORA (Cys-1), (Cys-2→Ala)-PORA (Cys-2), (Cys-3→Ala)-PORA (Cys-3), and (Cys-4→Ala)-PORA (Cys-4) before (A) and after (B) flashing the samples. (C and D) Pigment fluorescence analysis of the same POR-pigment complexes as those shown in A and B, respectively. (E and F) Western blots of proteins corresponding to those in A and B, respectively, after SDS/PAGE. Note the artificial migration behavior of PORB (PORB*) relative to PORA, which is caused by limited proteolysis of the enzyme occurring during sample handling.

Upon exposure to a 1-ms white light flash, PORB:(Cys-1→Ala)-PORA and PORB:(Cys-2→Ala)-PORA supracomplexes dissociated into fluorescing PORA-pigment and PORB-pigment subunits (Fig. 4 B and D). In the case of the PORB:(Cys-2→Ala)-PORA supracomplex, this dissociation seemed incomplete, and small amounts of the established higher molecular mass complex remained detectable. Strikingly, no comparable dissociation occurred for the PORB:(Cys-3→Ala)-PORA higher molecular mass complex, and only trace amounts of free PORA-pigment and PORB-pigment subunits were observed (Fig. 4 B and D). In the case of (Cys-4→Ala)-PORA, which had not formed larger complexes (Fig. 4 A and C), only fluorescing PORA-pigment and PORB-pigment subunits lighted up on the gels (Fig. 4 B and D).

Interestingly, slight differences became apparent in the migration behavior of the resolved PORB–pigment complex. PORB was present in slightly faster and slightly slower migrating bands, designated PORB-II and PORB-I, respectively (Fig. 4 B and D). Whereas PORB-I was abundant in incubation mixtures containing (Cys-3→Ala)-PORA and (Cys-4→Ala)-PORA, PORB-II was present only in mixtures containing (Cys-1→Ala)-PORA and (Cys-2→Ala)-PORA. HPLC analyses performed with pigments extracted from these two different PORB protein bands identified PORB-II as containing Chlide a and PORB-I as containing Pchlide a. Pigments extracted from the PORA protein band in all four cases provided Pchlide b as the only detectable pigment. When we extracted pigments from the nondissociable PORB:(Cys-3→Ala)-PORA high molecular mass complex, Pchlide a, Pchlide b, and Chlide a were resolved in a stoichiometry ≈1:2.5:0.01. The fact that no Chlide b was detectable in all of our experiments suggested that energy transfer from Pchlide b to Pchlide a eliminated the possibility of Pchlide b photoreduction.

Role of PORA in Photoprotection.

For directly testing the previously proposed photoprotective role of LHPP (15), consisting of the PORA and PORB, with their corresponding Pchlide pigments, NADPH and membrane lipids, we measured singlet oxygen (¹O₂) production in vitro. Lipid-containing PORA:PORB supracomplexes were reconstituted as described in Fig. 4, and the method used the DanePy reagent (34, 35), which is a dansyl-based ¹O₂ sensor undergoing quenching of its green fluorescence upon reacting with singlet oxygen.

Fig. 5 shows time courses of ¹O₂ evolution for the different (CysX→Ala)-PORA-Pchlide b-NADPH:PORB-Pchlide a-NADPH:lipid supracomplexes and unassembled ternary complexes. For assays containing (Cys-1→Ala)-PORA- and (Cys-2→Ala)-PORA, no ¹O₂ production was detectable. For assays consisting of nonphotodissociable (Cys-3→Ala)-PORA:PORB complexes, low rates of ¹O₂ production were measured that reached a plateau after 10 min. By contrast, ¹O₂ production was linear for at least 1 h and yielded ≈10-fold higher final ¹O₂ levels in assays consisting of unassembled (Cys-4→Ala)-PORA and PORB. The linear relationship between ¹O₂ production and time excluded the possibility that the nitroxide radicals formed from DanePy were partly reduced and transformed into inactive states in the samples (34).

Fig. 5.

¹O₂ production by reconstituted (CysX→Ala)-PORA:PORB:lipid complexes. Lipid-containing PORA:PORB supracomplexes containing the different (CysX→Ala)-PORA proteins were reconstituted as described in Fig. 3. ¹O₂ production was measured with the DanePy reagent after successive 1-ms flashes of nonsaturating white light, each applied at 1-min intervals. ○, (Cys-1→Ala)-PORA; ●, (Cys-2→Ala)-PORA; □, (Cys-3→Ala)-PORA; ■, (Cys-4→Ala)-PORA.

Discussion

In the present work, a structure–function study was performed for the PORA of barley. PORA represents the negatively light-regulated POR enzyme that rapidly disappears from etiolated plants upon illumination. Its expression and role is confined to etiolated plants, where it operates in light harvesting, energy transfer, and photoprotection during greening (36). Both aspects are illustrated in the present work. Using an in vitro-mutagenesis approach, two distinctive Pchlide binding sites were identified in the PORA. Two of the four evolutionarily conserved Cys residues, namely Cys-268/200 (operationally defined as Cys-3) and Cys-295/227 (operationally defined as Cys-4) were identified as participating in the establishment of the photoactive enzyme state and LHPP assembly, respectively. In the reconstituted LHPP complex, Pchlide b bound with NADPH to the PORA was involved in energy transfer to PORB-bound Pchlide a. By virtue of this mechanism the risk was kept low that triplet states of Pchlides a and b accumulated and provoked singlet oxygen production. Carotenoids present in the prolamellar body (37) may provide an additional mechanism to quench both excited singlet and triplet states of Pchlide and Chlide in etiolated plants undergoing greening.

Sequence alignments show that Cys-268/200 (Cys-3) is adjacent to the NADPH binding site. The overall sequence, which is defined as YKDSKVC in helix α–F, is identical in the PORA and PORB and is highly conserved between different POR enzymes, including synPOR (Fig. 1A) and pea POR (21). In the case of pea POR, the conserved Tyr and Lys residues have documented roles in NADPH and Pchlide binding. Lebedev et al. (28) reported that substrate and cosubstrate binding to POR occurs in two steps: an initial loose association of Pchlide and NADPH and a subsequent sort of “induced fit” that gives rise to the photoactive enzyme state. In the structural model depicted in Fig. 1, the YxxxK motif, comprising the universally conserved Tyr and Lys residues and the adjacent Cys-3, is readily accessible to its ligands. An as-yet-undetermined POR conformational change then is supposed to lead to the tight coordination of all reactants (8, 9, 28).

Cys-295/227 (Cys-4) constitutes a second, low-affinity Pchlide b binding site. We observed that Pchlide b bound to Cys-4 participates in PORA:PORB interactions. Replacement of Cys-4 by an Ala residue reduced by half the amount of bound pigment per PORA enzyme monomer and gave rise to assembly-incompetent PORA molecules.

Both Cys-3 and Cys-4 are involved in energy transfer. This is apparent from the lack of Pchlide-650/657 in assays containing the (Cys-3→Ala)-PORA and (Cys-4→Ala)-PORA mutant proteins. In either case, no photoactive Pchlide-650/657 was detectable by low-temperature fluorescence spectroscopy. Instead, bright pigment peaks appeared emitting at 630–635 nm. (Cys-3→Ala)-PORA established higher molecular mass complexes similar in size to those formed with WT PORA and the (Cys-1→Ala)-PORA and (Cys-2→Ala)-PORA. But these (Cys-3→Ala)-PORA-containing complexes were largely photoinactive and nondissociable upon treatment with flashes of white light. Trace amounts of Chlide a were produced. Spectroscopic evidence was obtained for a route of chlorophyll a synthesis not involving Pchlide-650/657 but depending on a Pchlide species fluorescing at 642 nm (Pchlide-F642). A similar Pchlide species had been detected by Lebedev et al. (32) using the det340/cop1 mutant of Arabidopsis thaliana that is largely depleted of PORA protein because of constitutive activation of phytochrome signal transduction depressing porA transcription. Lebedev et al. observed that etiolated det340/cop1 seedlings were especially light-sensitive (32). Similar results were reproduced in this work for PORB:(Cys-3→Ala)-PORA supracomplexes reconstituted with membrane lipids. We interpret our result as evidence for a partial protection of PORB by (Cys-3→Ala)-PORA from excess light.

A major breakthrough toward the understanding of PORA's presumed photoprotective role during greening was provided by the experiment described in Fig. 5. The measured rates of ¹O₂ production were extremely low for functional LHPP complexes. Presumably because of energy transfer from Pchlide b to Pchlide a and subsequent Pchlide a to Chlide a reduction, no significant levels of ¹O₂ were produced. By contrast, perturbation of LHPP's normal function, such as assembly of (Cys-3→Ala)-PORA with PORB into larger, nonlight-dissociable complexes, led to a marked increase in the rate of ¹O₂ evolution. Even more deleterious effects were observed for assays containing unassembled, but lipid-associated (Cys-4→Ala)-PORA and PORB. In this case, probably most of the incoming photons drove singlet oxygen formation. Although the atomic details of LHPP are not known yet, our work defines a photoprotective role of PORA during greening.

Materials and Methods

Primers.

Primers used were: Oligo-S, 5′-pCCGCGAGACCCACCCTTGGAGGCTCCAGATTTATC-3′; M1 (Cys-106→Ala)-PORA, 5′-pCACGTCGTCATGGCGGCGCGCGACTTCCTCAAG-3′; M2 (Cys-158→Ala)-PORA, 5′-pCTGGATGTGCTGGTCGCGAACGCCGCCATCTAC-3′; M3 (Cys-268→Ala)-PORA, 5′-pAAGGACAGCAAGGTGGCCACATGCTGACCATG-3′; and M4 (Cys-295→Ala)-PORA, 5′-pTCGCTCTACCCGGGCGCCATCGCCACG-GGG-3′.

Production of (CysX→Ala)PORA Mutant Proteins.

DNAs encoding the different (Cys→Ala)-PORA proteins lacking their transit peptides for plastid import were generated with clone A7 (12) as template and the Promega GeneEditor in vitro site-directed mutagenesis kit. The following primer combinations were used: Oligo-S plus M1, Oligo-S plus M2, Oligo-S plus M3, and Oligo-S plus M4. The resulting DNAs were subcloned into pQE30 vector (Qiagen) and transformed into E. coli strain XL1-Blue. Details on protein expression and purification are provided in SI Text. Alternatively, ³⁵S methionine-labeled POR proteins were produced by coupled transcription/translation of respective clones in wheat germ lysates (17).

Assembly Assay of LHPP.

Equimolar amounts of the different reconstituted (CysX→Ala)-PORA-Pchlide b-NADPH and PORB-Pchlide a-NADPH ternary complexes were mixed and incubated in the dark for 15 min. One aliquot of the reaction mixtures was immediately precipitated with trichloroacetic acid and used for radioactivity measurements done with a liquid scintillation counter. Another aliquot was subjected to gel filtration on Superose 6 (Amersham Pharmacia Biotech) equilibrated in assay buffer containing 350 mM sucrose. Individual fractions were harvested, and aliquots were withdrawn for radioactivity measurements. Alternatively, nonradiolabeled proteins were used and detected by Western blotting, using POR-specific antiserum (33). In either case, appropriate fractions were pooled and protein-precipitated with trichloroacetic acid, processed with acetone, ethanol, and diethyl ether, and resolved on 10–20% polyacrylamide gradients containing SDS (38). After electrophoresis, ³⁵S-PORA:PORB higher molecular weight complexes and nonassembled ³⁵S-PORA, ³⁵S-PORB, and ³⁵S-PORA derivatives were visualized by autoradiography or, in experiments where nonradiolabeled proteins had been used, by Western blotting.

Singlet Oxygen Measurements.

¹O₂ production was measured by using either the electrophoretically resolved PORA:PORB:lipid complexes or nonfractionated complexes and the DanePy method developed by Kálei et al. (34, 35). Fluorescence emission spectroscopy was carried out at an excitation wavelength of 330 nm and collecting fluorescence emission between 515 and 550 nm and above 650 nm in a Life Sciences spectrometer LS50 (PerkinElmer).

Pigment Measurements.

HPLC was performed on either C18 reverse-phase (RP) silica gel columns (Macherey-Nagel; 250 × 4.6 mm, Nucleosil ODS 5 μm) or C30 RP columns (YMC; 250 × 4.6 mm, 5 μm), using established procedures and a Varian ProStar model 410 apparatus, ProStar model 240 pump, and ProStar 330 photodiode array detector (16, 17). In some experiments, Chlides a and b were separated on RP18 Gromsil columns (Grom), using a gradient of 100% acetone, applied for 3 min, and 60% acetone/40% sodium acetate-supplemented water, pH 6.5, reached within 20 min (39). Low-temperature luminescence spectroscopy was performed at 77 K at excitation wavelengths of either 440 or 470 nm in the LS50 spectrometer (see above) (32).