Photoactive Protochlorophyllide-Enzyme Complexes Reconstituted with PORA, PORB and PORC Proteins of A. thaliana: Fluorescence and Catalytic Properties

Abstract

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.

Photosynthetic activity of variegated leaves of Coleus × hybridus hort. cultivars characterised by chlorophyll fluorescence techniques

“…In the creamy-yellow and pink sectors, the Chl a/b ratio was markedly lower and within the small pool of Chls, Chl b was more abundant than Chl a, which is unusual (Kim et al 2009). The presence of protochlorophyllides may be an explanation, because they have the absorbance spectra similar to Chl b (Belyayeva and Litvin 2011, Gabruk et al 2015).…”

Three-Dimensional Visualization of the Tubular-Lamellar Transformation of the Internal Plastid Membrane Network during Runner Bean Chloroplast Biogenesis

“…After 1 h of illumination, the 653-nm band completely disappeared and a new red-shifted band at ;679 nm was observed. This indicates a decline of Pchlide associated with LPOR complex, despite the significant band, still present, corresponding to free Pchlide (Schoefs and Franck, 2008;Gabruk et al, 2015). The corresponding excitation spectra, reflecting (Figure 9B), showed a Chlide/chlorophyll a related band at ;440 nm (Gabruk et al, 2015), suggesting energy transfer to Chlide/chlorophyll a species only.…”

“…This indicates a decline of Pchlide associated with LPOR complex, despite the significant band, still present, corresponding to free Pchlide (Schoefs and Franck, 2008;Gabruk et al, 2015). The corresponding excitation spectra, reflecting (Figure 9B), showed a Chlide/chlorophyll a related band at ;440 nm (Gabruk et al, 2015), suggesting energy transfer to Chlide/chlorophyll a species only. Simultaneous structural investigation by mild denaturing electrophoresis ( Figure 9D) and immunodetection ( Figure 9E) did not reveal the presence of either CP complexes or proteins associated with PSII.…”

Plant Protochlorophyllide Oxidoreductases A and B

“…Very recently, reconstituted enzyme complexes of POR A, POR B, and POR C from A. thaliana were studied with respect to their low temperature fluorescence spectra and catalytic activity (26). Whereas the fluorescence spectra indicate formation of highly aggregated POR complexes under the experimental conditions used, POR A and POR B did not differ in their catalytic activity.…”

“…Thus far POR A and POR B have been mainly examined in comparative studies with respect to their function in light-harvesting complexes (19,20,26). Although POR B is the key enzyme in chlorophyll synthesis of light-adapted, green plants, details of the enzyme kinetics and catalytic mechanism are still unclear.…”