Joanna Grzyb

The xanthophyll cycle - molecular mechanism and physiological significance

The light-dependent, cyclic changes of xanthophyll pigments: violaxanthin, antheraxanthin and zeaxanthin, called the xanthophyll cycle, have been known for about fifty years. This process was characterised for higher plants, several fern and moss species and in some algal groups. Two enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE), belonging to the lipocalin protein family, are engaged in the xanthophyll cycle. VDE requires for its activity ascorbic acid and reversed hexagonal structure formed by monogalactosyldiacylglycerol. ZE, postulated to be a flavoprotein, has not been purified yet and it is known from its gene sequence only. Zeaxanthin epoxidation is dependent on the reducing power of NADPH and presence of additional proteins.The xanthophyll cycle is postulated to play a role in many important physiological processes. Zeaxanthin, formed from violaxanthin under high light conditions, is thought to be a main photoprotector in autotrophic cells due to its ability to dissipate excess of absorbed light energy that can be measured as a non-photochemical quenching. In addition the zeaxanthin formation is important in protection of the thylakoid membranes against lipid peroxidation. Other postulated functions of the xanthophyll cycle, which include regulation of membrane physical properties, blue light reception and regulation of abscisic acid synthesis, are also discussed.

Empirical and computational design of iron-sulfur cluster proteins.

Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Light-dependent and light-independent protochlorophyllide oxidoreductases share similar sequence motifs —in silico studies

In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both in light-dependent protochlorophyllide oxidoreductase (LPOR) and in the L subunit of dark-operative (light-independent) protochlorophyllide oxidoreductases (DPOR). Amino acid residues of this motif shared similar physicochemical properties in both types of the enzymes. In the present paper, physicochemical properties of amino acid residues of this common motif, its spatial arrangement and a possible physiological role are being discussed. This is the first report when similarity between LPOR and DPOR, phylogenetically unrelated, but functionally redundant enzymes, is described.

Ferredoxin:NADP+ oxidoreductase as a target of Cd2+ inhibitory action--biochemical studies.

The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b(6)f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre - ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K(i) value calculated for research condition was 1.72 mM. FNR molecule can bind a large number of cadmium ions, as shown by the application of cadmium-selective electrode, but just one ion remains bound after dialysis. The effect of cadmium binding is significant disturbance in the electron transfer process from flavin adenine dinucleotide (FAD) to dibromothymoqinone, but less interference with the reduction of ferricyanide. However, it caused a strong inhibition of Fd reduction, indicating that Cd-induced changes in the FNR structure disrupt Fd binding. Additionally, the protonation of the thiol groups is shown to be of great importance in the inhibition process. A mechanism for cadmium-caused inhibition is proposed and discussed with respect to the in vitro and in vivo situation.

Visualization and characterization of prolamellar bodies with atomic force microscopy.

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.

Photoreduction of natural redox proteins by CdTe quantum dots is size-tunable and conjugation-independent

Colloidal CdTe quantum dots (QD) were able to reduce both heme and iron–sulfur cluster containing proteins. Reduction was depended on quantum dots size. CdTe nanocrystals emitting light with maximum at 550 nm (QD-550, r ∼ 1.5 nm) reduced both ferredoxin (Fd) and cytochrome c (Cyt c). The process was followed by UV/Vis absorption spectroscopy and steady state fluorescence spectroscopy. For CdTe emitting longer wavelength (QD-610 and QD-670) reduction of Fd was less efficient. QD emitting light with maximum at 750 nm (QD-750, r ∼ 3.3 nm) reduced only Cyt c. Reduction of proteins by QDs was photo-dependent and did not demand oxygen presence. As shown by gel filtration and fluorescence correlation spectroscopy, Fd formed complexes with QD while Cyt c did not bound steadily. The stable complex was not necessary for photoreduction, although might influence its kinetics. Time-resolved fluorescence studies showed than electron transfer rate depends on QD size and also is higher for QD–Cyt c when compared to QD–Fd. The mechanism of process was additionally explored by detailed analysis of QD–protein complex formation and by measurements of cyclic voltamperometry and zeta potential. These results open new possibilities in controlling of natural redox processes.

Effects of Amino and Thiol Group Reagents on the Ferredoxin:NADP+ Oxidoreductase Catalysed Reduction of Dibromothymoquinone

Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5′-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP+ oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 μM Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP+ oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.

Effect of Lipids on Violaxanthin and Diadinoxanthin De-Epoxidation

Violaxanthin (Vx) and diadinoxanthin (Ddx) de-epoxidation are light dependent steps in one of the most important photoprotecting processes called respectively violaxanthin and diadinoxanthin cycle. Violaxanthin cycle operates in vascular plants and many groups of algae while diadinoxanthin cycle is present in diatoms. In this study the influence of lipids on de-epoxidation of Vx and Ddx was investigated. In particular, the dependence between conversion of Vx into antheraxanthin and zeaxanthin as well as Ddx to diatoxanthin and the molecular dynamics of hydrophobic fraction of aggregates formed by inverted micelles, which are necessary for de-epoxidation, was studied. Thickness of the hydrophobic fraction of the aggregates, size of the inverted micelles, suggested by mathematical description of these structures and solubility of Vx and Ddx in various kind of lipids were the other tested parameters. Obtained results show that the rate of de-epoxidation is strongly dependent on physical/chemical properties of lipids. The key role for violaxanthin or diadinoxanthin de-epoxidase activation play non-bilayer lipids and the parameters of inverted micelles created by them, such as thickness, diameter and molecular dynamics of their hydrophobic core.