Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms[para]

Photochemistry and Photobiology, Aug 2003 by Mysliwa-Kurdziel, Beata, Amirjani, Mohammad R, Strzalka, Kazimierz, Sundqvist, Christer

ABSTRACT

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide^sub 650-656^ form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide^sub 650-656^ form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide^sub 628-633^ form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.

INTRODUCTION

In angiosperms the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) needs light. It is catalyzed by the enzyme-reduced nicotinamide adenine dinucleotide phosphate (NADPH)-protochlorophyllide oxidoreductase (POR; EC 1.3.1.33) in a light-driven reaction. The POR enzyme forms a ternary complex with its substrate Pchlide and cofactor NADPH. Three distinct isoforms of POR (PORA, PORB and PORC) have been identified in Arabidopsis.

Three sites on the Pchlide molecule arc of special importance for POR activity: the central metal atom, which should preferably be a magnesium atom; propionic side chain on the porphyrin ring D; and the structure of ring E (1). The Pchlide molecule fits into the substrate-binding pocket on the POR protein having the active site enabling the hydride transfer from NADPH located at the bottom. Conserved Tyr and Lys residues in POR contribute to the reaction by proton transfer (2).

The photobiochemical reaction of POR depends on the [pi] electron system of the macrocycle during excitation by light. The complex spectroscopic properties of Pchlide in dark-grown leaves indicate different interactions with the surroundings of the [pi] electron system of Pchlide. Four "universal" Pchlide forms have been identified, having fluorescence emission bands around 633, 640, 656 and 670 nm (3). Differences in their relative fluorescence intensity and position of maxima found in different species can be mainly due to varying interactions of the Pchlide molecules with each other and with POR (4).

The Pchlide^sub 650-636^ form is regarded as a highly aggregated complex of Pchlide, NADPH and POR (5,6), mainly localized to the prolamellar body (PLB) (7). The Pchlide^sub 638-642^ is also bound to POR, and as the Pchlide^sub 650-656^, it can be transformed to Chlide by millisecond flash irradiation. The short-wavelength form of Pchlide (Pchlide^sub 628-633^) is not transformed to Chlide by millisecond flashes, but organisms rich in this form can slowly become green under continuous light (8,9). The short-wavelength form is monomeric and more abundant in the prothylakoids than in the PLB (10). In POR-overexpressing wild type or cop1-18 mutant of Arahidopsis, the ratio of short- to long-wavelength Pchlide decreased (11,12), confirming the long-wavelength Pchlide form as POR bound. The presence of a fluorescence peak at 656 nm strongly correlates with the presence of regular PLB (11,13), and circular dichroism indicates the existence of aggregates (14).

POR seems to bind only one molecule of Pchlide to its active site. Still, the pigment is regarded as being present in a dimeric form (15) in plants, and the POR molecule is regarded as forming aggregates together with the Pchlide pigment (6,14). Confirming earlier suggestions (16,17), Klement et al. (18) showed the influence of the lipid composition surrounding the enzyme on the spectral properties of POR-bound Pchlide.

Fluorescence lifetime measurements can be used to further characterize the different pigment forms to understand better their molecular surroundings. Earlier measurements indicate that photoactive and nonphotoactive Pchlide have different lifetimes (19). Two lifetime components were also found in time-resolved fluorescence measurements of etioplast membranes (20). Using etioplast preparations and measuring lifetimes both at 287 and 103 K, it was shown that the Pchlide^sub 650-656^ had a complex character of its fluorescence decay, with a fast component of 0.25 ns and a slower component of about 2 ns (21). Furthermore, lifetime components of 2 and 5.5-6.0 ns were characteristic for the Pchlide^sub 638-642^ form and the Pchlide^sub 628-633^, respectively.