The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize ( Zea mays ) Eva Selstam 1 , Jenny Schelin 1 , Tony Brain 2 and W. Patrick Williams 2 1 Umea ˚ Plant Science Center, Department of Plant Physiology, University of Umea ˚ , Sweden; 2 Life Sciences Division, King’s College, University of London, UK Prolamellar bodies (PLB) contain two photochemically active forms of the enzyme protochlorophyllide oxido- reductase POR-PChlide 640 and POR-PChlide 650 (the spec- tral forms of POR-Chlide complexes with absorption maxima at the indicated wavelengths). Resuspension of maize PLB in media with a pH below 6.8 leads to a rapid conversion of POR-PChlide 650 to POR-PChlide 640 and a dramatic re-organization of the PLB membrane system. In the absence of excess NADPH, the absorption maximum of the POR complex undergoes a further shift to about 635 nm. This latter shift is reversible on the re-addition of NADPH with a half-saturation value of about 0.25 m M NADPH for POR-PChlide 640 reformation. The disappearance of POR- PChlide 650 and the reorganization of the PLB, however, are irreversible. Restoration of low-pH treated PLB to pH 7.5 leads to a further breakdown down of the PLB membrane and no reformation of POR-PChlide 650 . Related spectral changes are seen in PLB aged at room temperature at pH 7.5 in NADPH-free assay medium. The reformation of POR- PChlide 650 in this system is readily reversible on re-addition of NADPH with a half-saturation value about 1.0 l M . Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide 650 ,membrane organization and NADPH binding. The low-pH driven spectral changes seen in maize PLB are shown to be accelerated by adenosine AMP, ADP and ATP. The significance of this is discussed in terms of current suggestions of the possible involvement of phosphorylation (or adenylation) in changes in the aggregational state of the POR complex. Keywords: protochlorophyllide oxidoreductase; prolamellar body; protochlorophyllide; oxidoreductase; chlorophyllide. Plant prolamellar bodies (PLB) found in the etioplasts of dark-grown (etiolated) seedlings, are the precursors of the chloroplast thylakoid membrane. The PLB membrane is dominated by the presence of a single protein species, protochlorophyllide oxidoreductase (EC 1.3.1.33) (POR) that catalyses the light-driven, NADPH-dependent reduc- tion of protochlorophyllide (PChlide) to chlorophyllide (Chlide). Analyses of the absorption spectrum of PLB [1] and low-temperature fluorescence spectra of etioplast inner membrane preparations (EPIM) and PLB [2], indicate the presence of three major pools of PChlide; a nonphotocon- vertible form PChlide 628)633 and two photoconvertible forms PChlide 640)645 and PChlide 650)657 . The suffix numbers relate to the wavelengths of the absorption and emission maxima, respectively. To emphasize the fact that the two photoconvertible forms are bound to POR, they will be referredtoheretoasPOR-PChlide 640 and POR-PChlide 650 . Under in vivo conditions, exposure of etioplasts to a flash of bright white light leads to a conversion of the photo- convertible PChlide pigments to Chlide resulting in a rapid shift of the main absorption maximum from 650 nm, initially to about 678 nm and then to 684 nm. Over a period of about 20 min, this absorption maximum shifts back to 672 nm. This latter shift, referred to as the Shibata shift [3], is attributed to the release of Chlide from POR. This release is accompanied by extensive changes both in the composition and morphology of the PLB eventually leading to its conversion to the chloroplast thylakoid membrane system. Isolated PLB show a similar pattern of spectroscopic changes immediately following illumination. The presence of excess NADPH, however, is required to ensure the replacement of the NADP + by NADPH and drive the absorption peak shift from 678 to 684 nm [4,5]. Under these conditions, no Shibata shift occurs and the PLB lack the ability to undergo the compositional and morphological changes seen in vivo. The relationship between the two photoconvertible forms of POR has been the subject of much discussion. A number of lines of evidence suggest that POR-PChlide 640 and POR- PChlide 650 are the less and more aggregated forms, respect- ively, of the same enzyme [1,6–8] and Ryberg, Sundqvist and their coworkers [9–11] have recently reported results suggesting that this aggregation may be favoured by POR phosphorylation. The idea of a possible phosphorylation Correspondence to W. P. Williams, Life Sciences Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN. Fax: + 44 20 7848 4450, Tel.: + 44 20 7848 4433, E-mail: patrick.williams@kcl.ac.uk Abbreviations: Chlide, chlorophyllide; PChlide, protochlorophyllide; PLB, prolamellar body; POR, protochlorophyllide oxidoreductase; POR-PChlide 635 ,POR-PChlide 640 , POR-PChlide 650 ,POR- Chlide 676)677 and POR-Chlide 684 , spectral forms of POR-PChlide or POR-Chlide complexes with absorption maxima at the indicated wavelengths; EPIM, etioplast inner membrane preparations. (Received 20 December 2001, revised 15 March 2002, accepted 19 March 2002) Eur. J. Biochem. 269, 2336–2346 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02897.x step in the interconversion of different forms of POR can be traced back to an early series of experiments by Horton & Leech [14,15] in which the transformation of POR- PChlide 650 to a short-wavelength form with an absorption maximum around 630 nm in aged maize etioplasts was found to be reversed by the addition of ATP. However, Griffiths [16], working with water-lysed oat etioplasts, observed the formation of a similar species absorbing at 633 nm that reconverted to POR-PChlide 650 on the addition of NADPH but was unaffected by the addition of ATP alone. Similar results were obtained by Brodersen [17] working with aged barley PLB. Current suggestions on the involvement of POR-phos- phorylation are centred around a series of more recent reports. Wiktorsson et al. [9] reported that the reformation of photoactive PChlide species in preilluminated etioplast inner membrane (EPIM) preparations suspended in low pH media is favoured by ATP. They also found the blue shift in the absorption maximum of the photoconverted enzyme occurring under these conditions to be inhibited by ATP and by the protein phosphatase inhibitor NaF. On the basis of this study, and subsequent studies on the action of protein kinase and protein phosphate inhibitors [10,11], POR-PChlide 650 was suggested to be an aggregated form of a phosphorylated (or possibly adenylated) ternary complex of POR, its substrate PChlide, and NADPH. Dephospho- rylation of the photoconverted form of this complex by an endogenous phosphatase, it was further suggested, leads to a disaggregation of POR and a dissociation of Chlide from the POR complex, giving rise to the Shibata shift. In this paper, the resuspension of maize PLB in media with a pH below about pH 6.8, is shown to lead to a rapid conversion of POR-PChlide 650 to POR-PChlide 640 .These changes, which take place over an extremely narrow pH range, are shown to be accompanied by marked decreases in the ability of POR to bind NADPH and a rapid disassembly of the PLB. The pH-driven spectral changes are compared to those seen in aged PLB. The effects of adenosine, AMP, ADP, ATP and NaF on the pH-driven changes are studied and discussed in terms of their relevance to the POR phosphorylation model. MATERIALS AND METHODS PLB isolation Maize seedlings (Zea mays L. cv. Apache) were grown in the dark for 9 days at 24 °C in a peat-soil mixture containing fertiliser. PLB, isolated according to the proce- dure of Widel-Wigge & Selstam [12], were stored at )20 or )70 °Cin1.3 M sucrose, 50 m M KCl, 1 m M MgCl 2 ,1m M EDTA, 0.3 m M NADPH, 20 m M Tricine, 10 m M Hepes, adjusted to pH 7.5 with KOH. All experiments, unless otherwise specified, were carried out at room temperature on PLB freshly resuspended in assay medium containing 250 m M sucrose, 50 m M KCl, 1 m M MgCl 2 ,1m M EDTA and 30 m M Hepes adjusted to pH 6.5 or pH 7.5. The results reported in this paper were normally based on measurements performed on at least three different PLB preparations. Minor differences in the rates of the spectral and structural changes were observed between different preparations but the overall pattern of changes was extremely consistent. Absorption and fluorescence spectrophotometry Aliquots (50 lL) of freshly thawed samples of PLB containing  200 lg protein, were thoroughly washed in cold assay medium at pH 7.5 to remove excess NADPH. The washed pellet was then re-suspended in 1.0 mL of test assay medium. Absorption spectra were normally measured using a Shimadzu MPS 2000 spectrophotometer fitted with a cuvette holder close to the photomultiplier to reduce light scattering. A few measurements were made using a Philips PU8720 spectrophotometer and a computer-generated baseline used to minimize the effects of light scatter. Photoconversion was brought about by exposure of the sample to a defined number of flashes of bright white light delivered by a Sunpak, Softlite 2000 A (Tocad, Tokyo, Japan). When required, 40 lL samples were removed for low-temperature (77 °K) fluorescence emission measure- ments made using a FluoroMax-2 spectrofluorimeter (Instrument S.A. Inc. Edison, NJ, USA). Transmission electron microscopy (TEM) Samples were fixed in 2.5% gluteraldehyde in 100 m M cacodylate buffer, pH 7.4. They were then post-fixed with osmium tetroxide, embedded and sectioned. The sections, stainedin2%uranylacetatefollowedbyleadcitrate,were examined using a Philips EM301G electron microscope. RESULTS pH dependence of the spectral properties of POR pigment complexes Resuspension of maize PLB in low pH media results in a rapid irreversible change in spectral properties. The room temperature absorption and 77 °K fluorescence emission properties of maize PLB resuspended in assay medium at pH 7.5 and pH 6.5 are compared in Fig. 1. The spectral characteristics of maize PLB suspended at pH 7.5 are very similar to those previously reported [1,2,4]. Prior to photoconversion, the PLB were characterized by a broad absorption peak with a maximum at about 650 nm and a broad shoulder at around 640 nm, associated with POR-PChlide 650 and POR-PChlide 640 , respectively (Fig. 1A). Exposure to a single flash of bright white light results in formation of the corresponding Chlide derivatives and a shift in absorption maximum, in the presence of excess NADPH, to 684 nm. The 77 °K fluorescence emis- sion spectrum of the nonphotoconverted PLB is dominated by the 656-nm emission peak of POR-PChlide 650 (Fig. 1B). Some emission from the nonphotoconvertible species PChlide 628 is visible at about 630–635 nm but little or no emission is seen from POR-PChlide 640 , reflecting the efficient excitation energy transfer existing between this species and POR-PChlide 650 [13]. Samples photoconverted in the presence of excess NADPH are characterized by a maximum at 696 nm typical of the Chlide derivative of POR-PChlide 650 . The results for maize PLB resuspended at pH 6.5 are strikingly different. Under these conditions, there is a rapid conversion of POR-PChlide 650 to POR-PChlide 640 (Fig. 1C). Exposure to bright white light leads to the photoconversion of POR-PChlide 640 to its corresponding Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2337 Chlide derivative with an absorption maximum at about 675 nm (Fig. 1C). Corresponding changes are seen in low- temperature fluorescence emission (Fig. 1D). The main emission peak prior to photo-conversion is now centred at 648 nm. There is no sign of the 656 nm emission peak seen at pH 7.5, reflecting the fact that POR-PChlide 650 is completely converted to POR-PChlide 640 . Following photo- conversion, the emission peak shifts to 682 nm reflecting the formation of the Chlide derivative of POR-PChlide 640 . The presence of excess NADPH is an important factor at low pH. If PLB are resuspended in pH 6.5 assay medium in the absence of excess NADPH, the PChlide absorption maximum shifts to about 635 nm rather than 640 nm, as illustrated in Fig. 2. Under these conditions, there is minimal photoconversion of the sample on exposure to light. On addition of NADPH, however, the absorption maximum shifts back to 640 nm and photoconvertibility is restored. These changes are attributed to the conversion of photoconvertible POR-PChlide 640 to a nonphotoconverti- ble species POR-PChlide 635 lacking bound NADPH which rapidly reconverts to POR-PChlide 640 in the presence of added NADPH. POR-PChlide 635 has a 77 °K fluorescence emission peak at 640 nm of similar intensity to the 648 nm emission peak of POR-PChlide 640 (data not shown) clearly distinguishing it from PChlide 628 , which emits at 633 nm and remains nonphotoconverted even in the presence of excess NADPH. The pH-driven conversion of POR-PChlide 650 to POR- PChlide 640 (POR-PChlide 635 in the absence of excess NADPH) is very rapid, taking less than a minute at room temperature and is complete in less than 10 min even at 0 °C (Fig. 3). As illustrated in Fig. 4, the process is irreversible. Samples exposed to pH 6.5 were resuspended in pH 7.5 assay medium containing 3 m M NADPH. The PChlide absorption maximum, however, remained close to Fig. 2. Absorption spectra of maize. (a) PLB freshly resuspended sample in pH 6.5 assay medium lacking NADPH; (b) after exposure to one flash of bright white light; (c) the same sample plus 1 m M NADPH;(d)afterexposuretoasecondflashofbrightwhitelight(e) after a 60-s exposure to full room light. Fig. 1. Light-driven changes in maize PLB. (A) Room-temperature absorption changes at pH 7.5. (B) Absorption changes corresponding to low-temperature fluorescence emission changes. (C) Room-tem- perature absorption changes at pH 6.5. (D) Corresponding low-tem- perature fluorescence emission changes. All samples contained 1.0 m M NADPH. Fluorescence excitation wavelength was 440 nm. Solid lines and dashed lines correspond to nonphotoconverted and photocon- verted forms, respectively. Fig. 3. Plots of the time dependence of the blue-shift in the absorption maximum of PChlide following resuspension of PLB in assay medium pH 6.5. Measurements were made at 5 °C in the presence (j)andthe absence (d)of1.0m M NADPH. 2338 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002 640 nm even after 20 min incubation at room temperature. Multiple flashes of saturating white light were required for full photoconversion of the incubated sample and led to the formation of a Chlide peak centred at about 675 nm characteristic of the low pH form (cf. Figs 2 and 4). A potential complicating factor in these latter measure- ments is the tendency of POR-PChlide 635 to break down to yield a new PChlide absorption band with a maximum at 653 nm (PChlide 653 ). Traces of this species are detectable in the spectra shown in Fig. 4. This breakdown is more clearly illustrated in Fig. 5, which shows the effects of ageing on the absorption spectrum of maize PLB suspended in pH 6.5 assay medium in the absence of excess NADPH. PChlide 653 is easily distinguishable from POR-PChlide 650 as it is nonphotoconvertible and gives rise to no obvious low- temperature fluorescence. It is probably related to the species PChlide 647 , attributed to aggregated protochloro- phyllide, reported in dried etioplast membrane preparations [19]. Care was taken in all measurements to restrict the time of exposure of PLB samples to low pH in media lacking excess NADPH to ensure that PChlide 653 formation was avoided. The pH-dependence of the conversion of POR- PChlide 650 to POR-PChlide 640 /POR-PChlide 635 is reflected in the measurements of the pH dependence of the wavelengths of the absorption maxima of PChlide and Chlide shown in Fig. 6. In both cases, there is a dramatic reduction in wavelength maximum over a narrow pH range spanning about 0.5 pH units centred at about pH 6.9. Structural studies The formation of PLB is generally believed to be associated with the presence of POR-PChlide 650 [20]. TEM measure- ments were therefore carried out to determine whether or not loss of POR-PChlide 650 correlates with loss of PLB structure. Typical electron micrographs of PLB samples suspendedinpH7.5andpH6.5assaymediumare Fig. 4. Changes in absorption spectrum of maize PLB. (a) Spectrum of sample initially suspended in pH 6.5 assay medium containing 3 m M NADPH and then pelleted, resuspended and incubated for 20 min at room temperature in pH 7.5 assay medium containing 3 m M NADPH. The sample was then exposed to one (b), two (c) and four (d) flashes of bright white light followed by (e) a 60-s exposure to full room light. Fig. 5. Absorption spectra showing the formation of PChlide 653 in PLB suspended in pH 6.5 assay medium incubated at room temperature for (a) 22, (b) 37, (c) 46, (d) 67, (e) 80, (f) 100 and (g) 125 min. Fig. 6. Plots showing the pH dependence of the absorption maxima of (A) PChlide in nonphotoconverted PLB samples (B) Chlide formed after photoconversion by two flashes of bright white light measured 2 min after conversion. Measurements were made in the presence (j)andthe absence (d)of1.0m M NADPH. Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2339 presented in Fig. 7. At pH 7.5 (Fig. 7A), the majority of the PLB are in the form of highly ordered paracrystalline arrays based on networks of interconnected tubular tetrapodal membrane units forming a bicontinuous diamond cubic (Fd3m) lattice [21,22]. Resuspension at pH 6.5 (Fig. 7B), however, leads to complete loss of such ordered structures and their replacement by highly disordered arrays of entangled tubes. Parallel X-ray diffraction measurements (data not shown) confirmed that resuspension of PLB in low pH media leads to a complete loss of long-range order in the samples. This breakdown in PLB structure, like the conversion of the POR complex, is irreversible. There is no evidence of the reformation of organized PLB if the pH of the low pH sample is returned to pH 7.5 by the addition of small amounts of KOH. In contrast, the turbid PLB suspensions rapidly clarify and become optically clear, suggesting a further breakdown of the tubular structures into small vesicles. NADPH binding studies The formation of POR-PChlide 635 , as opposed to POR- PChlide 640 , in PLB samples resuspended in low pH assay medium in the absence of excess NADPH strongly suggests that POR-PChlide 640 , under these conditions at least, has a greatly reduced affinity for NADPH. The NADPH binding capability of POR-PChlide 635 /POR-PChlide 640 was estima- ted by measuring the NADPH dependence of the photo- conversion of PLB at pH 6.5. Plots of the extent of photoconversion in response to a single saturating flash, and to a series of such flashes separated by a dark time of 30 s, are presented in Fig. 8A. The response to a single flash reflects the position of the equilibrium existing between photoconvertible POR-PChlide 640 and nonphotoconverti- ble POR-PChlide 635 . The higher yield achieved by multiple flashes, indicates the re-establishment of this equilibrium in the dark period between flashes. The equilibrium between the two forms is also reflected in the measurements of the NADPH dependence of the red-shift in the PChlide absorption maximum shown in Fig. 8B. In both cases, half-saturation of these changes occurs at  0.25 m M NADPH, indicating that POR-PChlide 640 binds NADPH comparatively weakly at pH 6.5. Interestingly, even at high levels of NADPH, a single saturating flash was unable to photoconvert all the pigment present at low pH. The reasons for this are unclear. This contrasts strongly with the binding of NADPH to POR-PChlide 640 at pH 7.5. Attempts to remove NADPH from POR-PChlide 640 and/or POR-PChlide 650 by repeated Fig. 7. Typical electron micrographs of PLB samples suspended in assay medium at (A) pH 7.5 and (B) pH 6.5. Magnification bar 200 nm. Fig. 8. NADPH concentration dependence of the photoconversion of PChlide to Chlide of PLB suspended in pH 6.5 assay medium. (A) Changes in percentage photoconversion in response to one, two, and four saturating flashes of white light. (B) Variation of the wavelength of the absorption maximum of PChlide with NADPH concentration. 2340 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002 washing in cold NADPH-free media were unsuccessful, suggesting that NADPH is effectively irreversibly bound to both forms of the enzyme under these conditions. The alternative approach of first resuspending the PLB in low pH media to dissociate bound NADPH and then restoring the suspension to pH 7.5 by addition of small amounts of KOH, prior to re-addition of NADPH was attempted. This, however, invariably led to a breakdown of an appreciable fraction of the POR-PChlide 635 complex to form PChlide 653 , which interfered with the subsequent spectral analysis. If NADPH is rebound before the restoration of the pH to pH 7.5, relatively little PChlide 653 is formed indica- ting the ability of NADPH to stabilize the enzyme. The NADPH binding properties of the photoconverted enzyme at pH 7.5 were investigated using the red-shift in the Chlide absorption maximum accompanying the displace- ment of bound NADP + by NADPH [4,5]. The samples were first photoconverted in the absence of excess NADPH to form the NADP + -bound enzyme. The extent of the red- shift following the addition of different concentrations of NADPH was then used to estimate the efficiency of NADPH binding. The results of these measurements, performed at 5 °C to slow down other possible changes in the photoconverted enzyme, are shown in Fig. 9. In the absence of added NADPH, the wavelength of the absorp- tion maximum measured 10–20 s after photoconversion was at  680–681 nm falling to  677–676 nm after about 5 min. The full red-shift was obtained even if the addition of the NADPH was delayed until the wavelength had stabil- ized at this shorter wavelength, indicating that this initial decline is not linked to a loss of Chlide (Fig. 9A). Approximately 2 l M NADPH was sufficient to drive the full shift (Fig. 9B). This approach, unfortunately, cannot be adopted at low pH as the enzyme is essentially nonphoto- convertible in the absence of excess NADPH and the red- shift, if one exists at all, is negligibly small. Comparison with the effects of ageing A similar, but much slower, conversion of POR-PChlide 650 to shorter wavelength forms is seen in aged etioplasts and PLB [14–17]. Maize PLB prewashed in NADPH-free assay medium (pH 7.5) were aged in the dark at room tempera- ture for five hours. After this time,  50% of the POR- PChlide 650 was converted to a shorter wavelength form with an absorption maximum close to 635 nm. This species (referred to in the early literature as P-630) is nonphoto- convertible, but is converted to a photoconvertible form in the presence of NADPH It is clearly very closely related to, if not identical to, POR-PChlide 635 . If excess NADPH is not removed, POR-PChlide 650 is initially converted to POR- PChlide 640 , which then slowly converts to the shorter wavelength form. Following their dark incubation, the aged samples were exposed to room light for one minute to convert all the photoconvertible PChlide present to Chlide (Fig. 10A). NADPH was then added to the samples to convert the remaining nonphotoconvertible PChlide to a photoconvertible form. To check that the product was indeed photoconvertible, the PLB were re-exposed to room light (Fig. 10B). The original conversion of POR-PChlide 650 to POR-PChlide 635 , its reconversion to POR-PChlide 650 and its subsequent photoconversion to POR-Chlide 684 ,are all clearly visible in the difference spectra shown in Fig. 10(C,D). These changes are similar to those reported byBrodersen[17],whoworkedwithagedbarleyPLB. The NADPH dependence of the reformation process was estimated from measurements of the amounts of reformed POR-PChlide 650 available for photoconversion from differ- ence spectra of the type shown in Fig. 10D. The half- saturation value for NADPH binding to POR-PChlide 635 at pH 7.5 estimated on this basis is  1 l M (Fig. 11). In agreement with the findings of Griffiths [16] for water-lysed etioplasts, we found no requirement for ATP in these changes. TEM measurements (not shown) indicated a decrease in the overall degree of order of the PLB with ageing but no dramatic structural changes of the type seen on exposure to low pH. Re-addition of NADPH had no obvious effects on structure. Adenyl nucleotide and fluoride sensitivity Ryberg & Sundquist and their coworkers [9–11] have presented a number of lines of evidence suggesting the Fig. 9. Changes in the wavelength of the Chlide absorption maximum following photoconversion of washed PLB samples suspended in pH 7.5 assay medium. (A) Plots of the time dependence in samples contained no added NADPH (d), 2 l M NADPH added directly after photo- conversion (j), 2 l M NADPH (r)or1 l M NADPH (m)added7 min after photoconversion. (B) Plot showing the NADPH concentration dependence of the red shift in the wavelength maximum of Chlide following the addition of NADPH to PLB photoconverted in the absence of excess NADPH. Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2341 involvement of a kinase/phosphatase system in the POR system. One line of evidence of particular relevance to the present study is their observation that ATP inhibited the low pH-induced blue-shift in the wavelength of the low- temperature fluorescence maximum of Chlide seen in wheat EPIM photoconverted in the presence of excess NADPH [9]. They also demonstrated that ATP and NaF (presum- ably acting as phosphatase inhibitors) inhibited the loss of the long-wavelength form of Chlide following photocon- version of reformed phototransformable PChlide in this system. The results of measurements of the effects of ATP, ADP, AMP and adenosine on the corresponding low pH-induced blue-shift in the absorption maximum of maize PLB, made in the presence and absence of NaF, are presented in Fig. 12A. The measurements were made by adding small aliquots (70 lL) of POR-PChlide 650 suspended in assay medium at pH 7.5 to a much larger volume (1 mL) of pH 6.5 assay medium containing 1 m M NADPH, immedi- ately photoconverting the sample by exposure to a satur- ating flash of white light and then monitoring the changes in wavelength of the Chlide absorption maximum. All meas- urements were performed at 5 °C to reduce the rate of the pH-driven conversion between the long- and short-wave- length forms of the enzyme. Additions of NaF, adenyl nucleotides and adenosine were made 2 min after photo- conversion to ensure the formation of POR-Chlide 684 prior to their addition. Measurements were restricted to the changes seen directly after initial photoconversion as no reformation of photoconvertible PChlide occurs in the maize PLB system. There is, however, no obvious reason why the stability of the reformed pigment complex should differ from that originally present. Fig. 10. Regeneration of POR-PChlide 650 in PLB aged in NADPH- free assay medium at pH 7.5. (A) The initial sample (thin line); aged sample before (thick line) and after (medium line) exposure to light. (B) Illuminated sample before (thin line) and after (thick line) dark incubation with 50 l M NADPH and subsequent reillumination (medium line) (C) difference spectra showing conversion of POR- PChlide 650 to POR-PChlide 635 during ageing (thick line) and the photoconversion of remaining photo-transformable pigment (medium line) (D) difference spectra showing the regeneration of POR- PChlide 650 in the presence of NADPH and (thick line) its subsequent photoconversion on reillumination (medium line). Fig. 11. NADPH dependence of regeneration of POR-PChlide 650 from POR-PChlide 635 estimated from difference spectra of type shown in Fig. 10D. Fig. 12. Plots of the time dependence of the pH-driven blue-shift in the absorption maxima of (A) Chlide and (B) PChlide (measured at 5° and 0 °C, respectively) following resuspension in assay medium pH 6.5 of PLB initially suspended in assay medium pH 7.5. All samples contained 1.0 m M NADPH with either no other additions (j), 10 m M NaF alone (d). 5 m M ATP (h), 5 m M ADP (s), 5 m M AMP (e), or 5 m M adenosine (n) in the presence or absence of 10 m M NaF as indicated. 2342 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002 In contrast to the study on wheat EPIM [9], the addition of ATP (or adenosine and the other adenyl nucleotides) accelerated rather than inhibited the blue shift. An inhibi- tion was observed if both ATP and NaF were present. A similar inhibition, however, was also observed for ADP, AMP and adenosine under these conditions indicating that in maize PLB at least this inhibition is not ATP-specific. In contrast to the study on wheat EPIM, no significant difference was seen between the rate of the blue shift in the presence or absence of NaF alone. The NADPH-binding efficiency of POR-Chlide 684 ,at low pH is unknown. It is thus hard to disentangle the effects of a possible loss of bound NADPH (leading to a reversal of the NADPH-dependent red shift seen in Fig. 11) from those of a physical dissociation of Chlide and/or conformational changes associated with the pH-dependent conversion of the enzyme from a more aggregated long-wavelength form to a less-aggregated short-wavelength form. In an attempt to isolate the contribution of the pH-driven conformational changes from the other effects, a parallel study was carried out on the low-pH induced blue shift in the PChlide absorption maximum of nonphotoconverted POR- PChlide 650 . Here, the photoconvertibility of POR- PChlide 640 , the final product, indicates that both the pigment and NADPH remain bound. The rate of the changes in absorption for the nonpho- toconverted enzyme was faster than those for the photo- converted form, necessitating measurement at 0 °Cas opposed to 5 °C. However, the general pattern of the results, presented in Fig. 12B, is very similar to that for the photoconverted enzyme, indicating that it is the conform- ational changes that predominate in both cases. Minor differences were seen in the rates of change seen for the adenyl nucleotides in the presence of NaF, with ATP showing the greatest inhibitory effect. To simplify presen- tation, only those changes seen for ATP and NaF + ATP are shown in Fig. 12B. The effect of the presence of the adenylates on the ability of POR-PChlide 640 to undergo photoconversion was checked by comparing the efficiency of photoconversion of PLB samples containing excess (1 m M ) NADPH in the presence and the absence of 5 m M adenosine or the adenyl nucleotides. Little or no difference was observed, indicating that although they had a marked effect on the stability of POR-PChlide 650 , they had little effect on the final level of NADPH binding to POR- PChlide 640 . Control measurements indicated that the effects of adenosine and the adenyl nucleotides were limited to the low pH range. No significant changes on the absorption spectra of nonphotoconverted PLB containing POR- PChlide 650 , or PLB photoconverted in the presence of excess NADPH to form POR-Chlide 684 , were observed at pH 7.5. However, as shown in Fig. 13, changes were seen, if the measurements on the photoconverted enzyme were made in the absence of excess NADPH. Under these conditions, the absorption maximum of the freshly photo- converted Chlide shows the usual decline from  679 to 676–677 nm. Addition of ATP leads to a rapid decrease in the wavelength to 673–674 nm. If NADPH is then added, the red-shift associated with the replacement of NADP + by NADPH is not observed indicating that the pigment has already dissociated from the parent enzyme. Similar, but smaller, blue shifts were seen for ADP and AMP but no discernible shift with respect to the adenylate-free control was seen in the case of adenosine. In all cases, the results were uninfluenced by the presence or absence of NaF. DISCUSSION Different spectral forms of POR The sensitivity of the absorption and fluorescence spectra of PChlide and Chlide of PLB to POR organization is well documented. Griffiths and his coworkers [4,5] successfully used this sensitivity to establish the basic framework of relationships existing between the different ternary com- plexes formed between POR, PChlide/Chlide and NADP(H). The proposed relationship between the different POR complexes referred to in this paper, based on the generally accepted scheme of Oliver & Griffiths [5], is summarizedinFig.14. The relationship between spectral changes in POR and structural changes in PLB The existence of a correlation between the presence of the cubic membrane structure of the PLB and the presence of POR-PChlide 650 has long been recognized [20]. This rela- tionship is underlined by studies on the etioplasts of mutants such as the cop1mutantofArabadopisis [23] and the lip1 mutant of pea [24], which are both deficient in POR- PChlide 650 and have been shown to be characterized by a parallel deficiency in organized PLB. The ability of the PLB to form a bicontinuous cubic phase is linked to the high proportion of the nonbilayer forming lipid monogalactosyldiacylglycerol (MGDG) pre- sent in the membranes. MGDG normally accounts for  50 mol% of the membrane lipids in the PLB membrane [25]. The presence of such a high content of nonbilayer forming lipid, however, is a necessary but not a sufficient cause for cubic phase formation. Whilst cubic structures can be formed in model systems containing high proportions Fig. 13. Plots of the time dependence of the blue-shift in the absorption maxima of Chlide following photoconversion of washed PLB suspended in assay medium pH 7.5 containing no excess NADPH. Samples con- tained no additions (j), or 5 m M adenosine or adenyl nucleotide in the presence (d)or(s) absence of 10 m M NaF. Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2343 of MGDG, they are stable under only very limited ranges of composition and hydration [26]. The existence of stable cubic structure in the PLB is dependent on the membrane protein content where POR is by far the dominant component. The mechanism by which POR-PChlide 650 stabilizes the cubic structure of the PLB is not fully understood but probably reflects its accumulation in, and subsequent stabilization of, membrane regions of a local curvature important to the overall stability of the cubic phase. Conversion of POR-PChlide 650 to POR-PChlide 640 , possibly by disaggregation, removes these constraints destabilizing the cubic phase, resulting in the relaxation of the membrane into the planar configuration characteris- tic of the prothylakoid region of the etioplast membrane (within the intact etioplast, at least). In the absence of attached prothylakoids, PLB are unable to undergo such a reorganization, possibly accounting for their high pH sensitivity. Driving force for pH-dependent changes Parallel changes in the spectral properties of POR and structural properties of PLB similar to those studied in this paper have been reported to occur in salt-washed PLB [12,27]. The two phenomena are clearly related and strongly point to the importance of changes in the surface properties of the PLB membrane. The obvious candidates for the driving forces in the case of the pH-dependent changes are either changes in the ionization of the membrane lipid headgroups, leading to a destabilization of the lipid–protein interactions that normally stabilize the cubic structure of the membrane, and hence to a destabilization of POR- PChlide 650 , and/or changes in the ionization of groups associated with POR. These changes then lead to the destabilization of POR-PChlide 650 and membrane reorgan- ization. The suspension of total polar lipid extracts of chloroplast membranes, which have a very similar lipid composition to the PLB membrane, in low pH media favours membrane fusion and formation of nonlamellar structures. The pK a for this process is  pH 4.5 [28], indicating that it reflects the protonation of the acidic lipids present in the mixture. The critical pH for the changes reported in the present study is close to pH 7, suggesting that the initial changes are unlikely to be directly related to changes in lipid headgroup ionization. Our results can be explained by attributing the effects of low pH to a reduction in the strength of NADPH binding to POR-PChlide 650 that triggers its relaxation to POR- PChlide 640 /POR-PChlide 635 , which then destabilizes the cubic structure of the membrane. This reduced NADPH binding capability of POR is reflected in the large disparity in the half-saturation concentration for the restoration of POR photoconvertibility in low pH treated PLB, about 0.25 m M NADPH at pH 6.5 (Fig. 8B), as compared to  1 l M NADPH for the reformation of POR-PChlide 650 in PLB aged at pH 7.5 (Fig. 10). The importance of mem- brane morphology in these processes is underlined by the failure of added NADPH to reform POR-PChlide 650 in low pH-treated PLB on restoration to pH 7.5 (Fig. 4). This is almost certainly a direct reflection of PLB membrane disruption linked with the pH-cycling. Once the membrane has reorganized into a tubular form, there is essentially no way back to a cubic structure under these conditions, hence the lack of reformation of POR-PChlide 650 . Addition of low concentrations of NADPH to PLB aged at pH 7.5 which show only limited structural reorganization, in contrast, results in a rapid reconversion of POR-PChlide 635 to POR- PChlide 650 with no obvious effect on membrane organiza- tion. Adenylate-sensitivity of pH effects POR, in common with many NADPH-binding enzymes, contains the characteristic motif Gly-X-X-X-Gly-X-Gly associated with the bA-aA-bB binding domain known as the Rossmann fold [29]. The detailed organization of the NADPH-binding pocket has yet to be established for POR but it has been determined in other members of the short- chain dehydrogenase/reductase family [30]. In common with Rossmann folds in general, these sites contain two mononucleotide binding sites; one for the nicotinamide and one for the adenosine moiety [31]. 2¢-Adenyl nucleotides, of the type found in NADPH, and the 5¢-nucleotides used in this study, are both known to bind within the adenosine site of such folds and can act as Fig. 14. Model illustrating the relationship between the different POR-PChlide and POR-Chlide complexes studied in this paper. 2344 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002 inhibitors interfering with NAD(P) + binding [32,33]. A possible explanation of the acceleration of the low-pH driven spectral changes seen for the photoconverted and nonphotoconverted enzymes on addition of the adenyl nucleotides or adenosine (Fig. 12) is that these compounds are able to compete for the NADPH binding site under low pH conditions destabilizing the aggregated POR-PChlide 650 complex. Their effect on NADPH binding to POR, however, appears to be transitory as the efficiency of binding of NADPH to POR-Chlide 640 at low pH, is not significantly impaired by the presence of 5 m M ATP. In contrast to the present results, Wiktorsson et al.[9] working with wheat EPIM observed a reduction in the rate of the low pH-induced blue shift in the Chlide fluorescence maximum in the presence of ATP. This could in principle reflect a species difference or a difference in the nature of the preparations. Measurements of Grevby et al. [18] indicate the retention of significant levels of low-temperature fluor- escence emission from POR-PChlide 650 in wheat PLB incubated at pH 6.0 for 48 h at 0 °C. This suggests that wheat PLB may be less pH-sensitive than their maize counterparts. Given the strong connection existing between POR organization and membrane morphology seen in the present study, the use of EPIM with their increased scope for membrane organization would only serve to enhance these differences. Relation to POR phosphorylation ThemeasurementsonPLBagedatpH7.5shownin Figs 10,11, confirm the finding of Griffiths [16] and Brodersen [17], who both suggested that the formation of POR-PChlide 650 from pre-existing POR-PChlide 635 (P-630), is solely dependent upon the presence of NADPH and does not appear to require ATP. This contrasts the earlier studies of Horton & Leech on aged maize etioplasts [14,15] where this conversion appeared to be ATP-driven. The possibility that the preparations employed by Griffiths and ourselves have lost the putative kinase during the course of prepar- ation cannot be excluded, but that still leaves unanswered the question of how the addition of micromolar concentra- tions of NADPH suffice to drive the conversion of POR- PChlide 635 to POR-Chlide 650 in its absence. It is noteworthy that the preparations used in the earlier studies contained sufficient excess NADPH to allow NADP + /NADPH exchange in the photoconverted enzyme leading to the formation of POR-Chlide 685 [15] raising the possibility that these results might reflect an ATP-dependent perturbation in NADPH binding efficiency rather than a direct POR- phosphorylation step. Kovacheva et al. [11] have recently reported ADP to inhibit the blue shift of the fluorescence emission of the Chlide peak of phototransformed wheat EPIM prepar- ations from 695 nm to 680 nm. A small amount of reformed phototransformable PChlide with an emission peak at 655 nm was observed at the same time. This inhibition was not seen if ADP was replaced by ATP. The reformed PChlide formed under these conditions was nonphototrans- formable and emitted at 651 nm rather than 655 nm. In the same report, the protein kinase inhibitor, K252a, was observed to inhibit the reformation of nonphototransform- able PChlide and no phototransformable PChlide was formed following subsequent addition of ATP and NADPH. The presence of a 695-nm emission peak indicates that the samples forming phototransformable PChlide contained sufficient residual NADPH to allow NADP + / NADPH exchange in the photoconverted pigment and, presumably, in the reformed PChlide complex. The above results could thus reflect a role for an ADP-dependent kinase step in the initial loading of PChlide on to POR. The absence of any apparent ATP (or ADP) requirement for the regeneration of photoconvertible PChlide in model systems supplemented with exogenous pigment [34] or for the mobilization of endogenous pigment in isolated etioplast membranes [35] would, however, seem to argue against a need for such a kinase. Klement et al. [36], have recently reported the formation of a complex between pigment-free POR and Zn-protopheide a in the presence of etioplast lipids. Again, no phosphorylation step is involved in pigment loading. The precise role of any possible POR kinase therefore still remains unclear. The main line of evidence for the existence of a POR- phosphatase is the observation by Wiktorsson et al. [9] of an inhibition, by NaF and ATP, of the loss of the long- wavelength form of Chlide following photoconversion of reformed phototransformable PChlide in wheat EPIM. In agreement with these findings, we observed related inhibi- tions of the pH-driven blue shift in Chlide and PChlide absorption in the presence of ATP and NaF (Fig. 12). The presence of NaF clearly perturbs the PLB system in some way. However, it must be emphasized that these effects, in maize PLB at least, are only observed under low-tempera- ture conditions and when adenosine or adenyl nucleotides are present. The possibility of other explanations of these effects cannot therefore be excluded. CONCLUSIONS The present study emphasizes the close relationship existing between the local organization of the PLB membrane and the stability of different POR-pigment complexes. It dem- onstrates the extreme sensitivity of these complexes, in PLB at least, to small changes in pH. It also confirms the central role of NADPH in the reformation of POR-PChlide 650 in aged PLB and raises the question that the sensitivity of spectral changes to the presence of adenyl nucleotides may reflect their effects on NADPH binding rather than their effects on specific phosphorylation (or adenylation) steps. ACKNOWLEDGEMENT The support of the Swedish Natural Research Council is gratefully acknowledged. REFERENCES 1. Bo ¨ ddi, B., Lindsten, A., Ryberg, M. & Sundqvist, C. (1990) Photo-transformation of aggregated forms of protochlorophyllide in isolated etioplast inner membranes. Photochem. Photobiol. 52, 83–87. 2. Bo ¨ ddi, B., Ryberg, M. & Sundqvist, C. (1993) Analysis of the 77 K fluorescence emission and excitation spectra of isolated etioplast inner membranes. J. Photochem. Photobiol. B. Biol. 21, 125–133. 3. Shibata, K. (1957) Spectroscopic studies on chlorophyll formation in intact leaves. J. Biochem. 44, 147–153. Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2345 [...]... Aggregation of NADPH -protochlorophyllide oxidoreductase- pigment is favoured by protein phosphorylation Plant Physiol Biochem 34, 23–34 10 Ryberg, M., Kovacheva, S & Sundqvist, C (1998) Accumulation of photo-transformable protochlorophyllide is controlled by protein phosphorylation – a hypothesis In Photosynthesis: Mechanisms and Effects Vol IV (Garab, G., ed.) pp 3197–3202 Kluwer Academic, the Netherlands... 5¢-triphosphate on the Shibata shift and on associated structural changes in the conformation of the prolamellar body in isolated maize etioplasts Plant Physiol 55, 393–400 16 Griffiths, W.T (1974) Source of reducing equivalents for the in vitro synthesis of chlorophyll from protochlorophyll FEBS Lett 26, 301–304 17 Brodersen, P (1976) Factors affecting the photoconversion of protochlorophyllide to chlorophyllide... focusing of pigment-protein complexes solubilised from non-irradiated and irradiated prolamellar bodies Physiol Plant 85, 659–669 8 Wiktorsson, B., Engdahl, S., Zhong, L., Boddi, B., Ryberg, M ¨ & Sundqvist, C (1993) The effect of cross-linking of the subunits of NADPH -protochlorophyllide oxidoreductase on the aggregational state of protochlorophyllide Photosynthetica 29, 205–218 9 Wiktorsson, B., Ryberg,... 283–296 24 Seyyedi, M., Timko, M.P & Sundquist, C (1999) Protochlorophyllide, NADPH -protochlorophyllide oxidoreductase, and chlorophyll formation in the lip1 mutant of pea Physiol Plant 106, 344–354 25 Selstam, E & Sandelius, A (1984) A comparison between prolamellar bodies and prothylakoid membranes of etioplasts of dark-grown wheat concerning lipid and polypetide composition Plant Physiol 76, 1036–1040... Netherlands 11 Kovacheva, S., Ryberg, M & Sundqvist, C (2000) ATP/ADP and protein phodphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes Photosyn Res 64, 127–136 12 Widell-Wigge, A & Selstam, E (1990) Effects of salt wash on the structure of the prolamellar body membrane and the membrane binding of NADPH -protochlorophyllide oxidoreductase Physiol Plant 78, 315–323... Selstam, E & Lindblom, G (1985) Phase equilibria of mixtures of plant galactolipids The formation of a bicontinuous cubic phase Biochim Biophys Acta 812, 816–826 27 Grevby, C., Engdahl, S., Ryberg, M & Sundqvist, C (1989) Binding -properties of NADPH -protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and immobilized prolamellar bodies Physiol Plant 77, 493–503 28 Gounaris,... (1970) Energy transfer between protochlorophyllide molecules: evidence for multiple chromophores in the photoactive protochlorophyllide protein complex in vivo and in vitro J Mol Biol 48, 85–101 14 Horton, P & Leech, R.M (1972) The effect of ATP on photoconversion of protochlorophyllide into chlorophyllide in isolated etioplasts FEBS Lett 26, 277–280 15 Horton, P & Leech, R.M (1975) The effect of adenosine... chlorophyllide in etioplast membranes isolated from barley Photosynthetica 10, 33–39 18 Grevby, C., Ryberg, M & Sundqvist, C (1987) Transformation of photoinactive to photoactive protochlorophyllide in isolated prolamellar bodies of wheat (Triticum aestivum) exposed to low pH and ATP Physiol Plant 70, 155–162 19 Brouers, M & Sironval, C (1975) Restoration of a P657-647 form from P645-638 in extracts of. .. stages of chlorophyll (ide) synthesis in etioplast membrane preparations Biochem J 152, 623–635 35 Franck, F., Bereza, B & Boddi, B (1999) Protochlorophyllide NADP+ and protochlorophyllide- NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranes of dark-grown wheat Photosynth Res 59, 53–61 36 Klement, H., Oster U & Rudiger, W (2000) The influence of ¨ glycerol and. .. Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase Biochemistry 38, 11440–11447 33 Busch, K., Piehler, J & Fromm, H (2000) Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by plasmon resonance and fluorescence spectroscopy Biochemistry 39, 10110–10117 34 Griffiths, W.T (1975) Characterization of the . The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize ( Zea mays ) Eva. explanation of the acceleration of the low- pH driven spectral changes seen for the photoconverted and nonphotoconverted enzymes on addition of the adenyl nucleotides