Figure 6. Spectroscopic comparison of Lv-wt black and Lv-L91P green. Absorption top and vis-CD bottom spectra after reconstitution with Chl a and after reconstitution with Chl b A and B, respectively. Absorption spectra were normalized to 1 at the Q y maxima.
Figure 7. Emission maxima after illumination F were measured upon excitation at Soret band maximum and compared to the initial maximum before illumination F 0. Figure 8. Comparison between the structures of Lv-wt 15 panels A and B and Lv-L91P 26 panels C and D , focused on the solvent-accessible part of the Chl molecule in the cavity highlighted with a black ellipse.
The same view is reported in the four panels, with the polypeptide chains in gray cartoons protein surface in transparent gray in panels B and D and the Chls a constituting a dimer shown either in sticks A and C or in spheres B and D.
The chlorin macrocycle is colored with carbon atoms in dark green, the methine carbon 20 in yellow, nitrogen atoms in blue, oxygen atoms in red, and magnesium atoms in gray. Figure 9. The same view is reported in the two panels, with the Chls a shown in sticks. Such files may be downloaded by article for research use if there is a public use license linked to the relevant article, that license may permit other uses.
This work has been funded by a grant from the Deutsche Forschungsgemeinschaft to H. More by Daniel M. More by Alessandro Agostini. More by Anne-Christin Pohland. More by Mara Werwie. More by Elmar Jaenicke. More by Harald Paulsen. Cite this: ACS Omega , 4 , 5 , — ACS AuthorChoice. Article Views Altmetric -. Citations Abstract High Resolution Image. Since the first appearance of oxygenic photosynthesis about 2.
This molecule has been selected during evolution presumably because of its ability to absorb and pass on excitation energy during light-harvesting and to undergo light-driven charge separation in the photosynthetic reaction centers. The role of the phytyl has been scarcely investigated and mostly connected to anchoring the Chls to the transmembrane proteins to which they belong 11,12 and affecting their packing.
A class of Chl-binding complexes in which phytyls seem to play a pivotal role is that of the non-photoconvertible water-soluble chlorophyll proteins WSCP. These proteins, found in plants belonging to the Brassicaceae family, are quite distinct from the majority of the Chl-binding proteins.
WSCP is not involved in the photosynthetic processes, as it is not a membrane protein and is not even located in the chloroplast. High Resolution Image. A close inspection of the crystal structure of L. This hydrophobic interaction was assumed to be the driving force for triggering the tetrameric assembly of WSCP complexes, 15 since it was thought that in the absence of phytyl chains, no oligomerization of WSCP occurred.
These tetrameric Chlide—protein complexes exhibited similar spectroscopic properties as their phytyl-containing counterparts. The recently solved structure of the L91P mutant of Lv-wt Lv-L91P, see Table S1 , where leucine is replaced by proline in the LCPS affinity motif, revealed striking differences regarding the conformation of the phytyl chains in comparison with the wt complex Figure 2.
When L91 is changed to the less bulky P91, more space between the Chl macrocycles and the backbone is available, allowing a reorganization of the phytyl chains. Consequently, they move away from the Chl macrocycles. A similar effect was reported by Saer et al. Intriguingly, in Lv-L91P, the two phytyl chains within one open-sandwich dimer adopt different conformations, with only one of the two protruding from the Chl planes toward the center and thus breaking the symmetry of the Chl dimer Figure 2.
Due to the important role of the phytyl chains in stabilizing the WSCP complex, 21,24 the question arose whether the altered phytyl conformation in Lv-L91P-bound Chls has an impact on their photo- and heat stability. To address this question, we compared Lv-wt and its L91P mutant with regard to their photo- and heat stability. By comparing the results of the Chl-reconstituted Lv-wt mutants with those collected for Lv-wt reconstituted with Chlide, we were able to assess the structural role of the phytyl chain and its conformation on the WSCP stability.
Results and Discussion. To gain more insight into the role of the phytyl groups in stabilizing WSCP toward thermal degradation, Lv-wt reconstituted with the phytyl-free Chl derivatives Chlide a and Chlide b was investigated. To quantify the complex integrity of the different WSCPs upon heat exposure, we measured vis-circular dichroism CD spectra before and after a 5 min boiling treatment Figure 3.
CD spectra are good indicators for the complex integrity because they depend on both pigment—protein and pigment—pigment interactions. In agreement with a previous study on A. Table 1. In the case of Lv-wt, a crystallographic structure is available 15 by contrast to A. In the complex, each of the four phytyl chains is in van der Waals contact with a cluster of hydrophobic side chains constituted by L41, L, and W of the subunit binding the corresponding Chl and the side chains of the same three amino acids of the opposing WSCP subunit A with C and B with D, see Figure 1.
The contribution of the phytyl chains to these hydrophobic interactions in the core of WSCP seems to be necessary for the extraordinary stability of the Chl—protein complex, as the absence of the phytyl chains led to a severe loss of complex heat stability Figure 3.
However, Chlides lacking the phytyl chains are still able to support tetramerization of Lv-wt, 21 albeit at lower complex stability, whereas in the absence of any Chls, no tetramers form. As in Lv-L91P the phytyl conformation of the bound Chls is altered, 26 by changing the interactions between the phytyls and their surroundings, we were interested in determining whether the hydrophobic interactions exerted by these phytyls still stabilize the complexes against heat denaturation the way they do in the wt structure.
We conclude that the differences in the conformation of the phytyls in L91P, with respect to Lv-wt, do not seem to affect the heat stability of the complex.
In Lv-L91P, only two of the four phytyl chains, each from an opposing Chl dimer, point to the center of the complex. Inspection of the crystal structure of Lv-L91P reveals that each of the two protruding phytyls is in van der Waals contact with the side chains of L41, L, and W, similarly to the wt. Since apparently the side-chain length of the amino acid at position 91 strongly affects the conformation of the phytyl chains as evident from the L91P mutation , we extended this mutational study by investigating the effect of three more mutations with decreasing side-chain lengths valine, alanine, and glycine at position 91 L91V, L91A, and L91G, respectively; see Table S1 and Figure 5.
CD spectra have been recorded in the UV region to assess the secondary structure of the proteins, to verify if the mutations affected the overall fold of the complexes. This indicates that all mutated proteins bind Chls at the same Chl—protein stoichiometry, forming Chl dimers with an open-sandwich geometry. When Lv-L91P was reconstituted with Chl b , a significant variation in the relative intensities between the Soret and Q y region, as well as a 2 nm red shift of the Q y absorption was observed in comparison with Chl b Lv-wt Figure 6 B.
In addition, the maximum of the Soret band absorption in Chl b Lv-L91P was shifted by 3 nm to the blue. The vis-CD spectra show differences between L91P and Lv-wt with respect to the circular dichroism in both Q y and Soret bands of bound Chl b molecules.
As the CD signals in the Q y are attributed to the characteristic open-sandwich conformation of the Chls bound, 29,30 the observed alterations in Chl b Lv-L91P, with a more conservative CD signal, indicate a modified Chl—Chl interaction, presumably originating from a modified Chl—protein interaction.
This putatively altered Chl—Chl interaction in the case of Chl b bound to Lv-L91P has been correlated to the variation of the hydrogen-bond network involving the formyl group of Chl b by comparing the crystal structure of Chl b Lv-L91P with that of Lv-wt. In all of these mutated proteins, the residues in position 91 provide a hydrogen-bond donor like in Lv-wt and consistently they all exhibit nonconservative CD signals same as those of the wt see Figure S2.
The heat stability of these mutants was evaluated, similar to that for Lv-L91P. Intriguingly, the Chl a -reconstituted as compared with the Chl b -reconstituted complexes seem to be somewhat less stable, as their calculated losses were roughly twice as high. As the only difference between the two pigments is the presence of a formyl group in Chl b , it is tempting to attribute the observed difference to the capability of Chl b to establish an additional H-bond with its surrounding, with a consequent increase in the stability.
Congruently, it has been observed, in the case of the major light-harvesting complex LHCII, a plant antenna protein that binds both Chl a and b that the loss of Chls b affects its stability more than the loss of Chls a. In previous studies, we have shown that Chls bound to WSCP are accessible 33 and can efficiently photosensitize oxygen to singlet oxygen 1 O 2 21 but even so undergo only little photodynamic damage, 17,21,23 even in the absence of any protecting carotenoids.
The underlying photoprotective mechanism that we proposed 21 is based on the capability of the phytyl chains either to shield methine 20 of the Chl macrocycle, which has been shown to be prone to the reaction with 1 O 2 , 34,35 or to limit the interaction of the oxygen with the magnesium ion, decreasing the residency time of the singlet oxygen near the Chl macrocycle 36 and consequently decreasing its probability to react.
This mechanism has been proposed on the basis of the loss of photostability of the WSCP complex when Chlide a is bound in place of Chl a , similar to the loss of heat stability presented and discussed above Figure 3. The role of the phytyls in the photoprotection of WSCP-bound Chls, shielding the more reactive portions of the chlorin macrocycle, relies on the presence of the Chl phytyl chains with a specific localization in the complex.
Therefore, it is expected to be highly dependent on the phytyl conformations, as the phytyl chains are required to be in van der Waals contact with the portions of the macrocycle involved in the reaction with 1 O 2.
As the Lv-L91P complex has been shown to exhibit a strikingly different phytyl conformation Figure 2 , 26 we have tested whether in Lv-L91P the bound Chls are still protected against photodamage.
To address this question, photodynamic damage was monitored, as previously reported, 17,21,23 by measuring the decrease of fluorescence intensity upon photooxidation of the Chls. To address this question, photobleaching measurements with Lv mutants exhibiting different side-chain lengths in position 91 L91V, L91A, and L91G were performed under identical experimental conditions.
By comparing the different mutants L91P, L91V, L91A, and L91G with regard to their ability to protect the bound Chls from photodynamic damage, it clearly appears that the loss of photostability is gradual, exhibiting an inverse correlation to the size of the side chain of the residue in position 91 Figure 7 A and Table 1. This strongly suggests that the more the bulkiness of the amino acid side chain in position 91 is reduced in the series from L via V, A, P, to G , the more the phytyl chain will be displaced from its position in the wt structure, leading to a gradually decreased photostability.
Whereas Lv-wt complexes reconstituted with the two Chls have nearly identical photostability, the Chl b -reconstituted mutants show a slightly smaller loss of photostability when compared with the corresponding Chl a -containing complexes.
This difference does not seem to be affected by the capability of residue to provide a hydrogen-bond donor, as the L91P follows the same trend, and the difference in photostability seems to be greater for the variants with smaller side chains of residue Therefore, these results corroborate the hypothesis that the observed difference in the photostability has to be assigned to a difference in the space available for the phytyl chains to adopt their conformation.
Upon reorganization of the phytyl chains of the Chls, induced by the less bulky P91 residue see above , a channel appears to be formed providing access for the 1 O 2 to the methine 20 of the Chl macrocycles and to the magnesium ion as well.
This may explain why the Chls are not photoprotected any more upon exchanging leucine with proline at position We propose that in the series of amino acids in position 91 L, V, A, G, P analyzed in this study, the gradual loss in photostability is explained by the phytyl conformation being gradually altered upon the reduction of steric hindrance exerted from the amino acid side chain, leading to a gradually reduced protection of the chlorin ring that has to be ascribed to a less effective masking of the reactive portions of the macrocycle.
The modification of the available space inside the cavity, besides allowing for changes in the static structure of the phytyl chains see Figure 2 , in principle, could also cause an increase of the phytyl conformational freedom.
We compared the B-factors of the atoms of the Chls bound in the two structures Figure 9 , revealing a similar pattern with a certain degree of flexibility in the phytyl chains in comparison with the high rigidity in the localization of the atoms of the chlorins.
The phytyls in the L91P are slightly more flexible, a trait that could also have concurred to the loss of photoprotection of the chlorins. An alternative explanation for the dependence of the photostability of the bound pigments to mutations of L91 would be that the different amino acid side chains interact differently with 1 O 2.
Since all amino acids investigated in this study have an aliphatic side chain or a hydrogen atom in the case of glycine , their direct reaction with 1 O 2 can be excluded. Therefore, we conclude that photoprotection of the bound Chls in WSCP 21 depends not only on the presence of the phytyl chains but also on their correct localization, enforced by the protein surrounding, within the two adjacent Chl macrocycles in the open-sandwich dimer.
The biochemical and photophysical data described in this paper highlight that both phytyl—phytyl and phytyl—protein interactions in the WSCP core are involved in generating the unusual photo- and heat stability of WSCP that characterize these plant proteins, confirming that phytyl chains play a central role in the WSCP complex. In particular, we have shown that the remarkable heat stability of WSCP depends on the presence of its Chl phytyl chains that interact with one another, with both chlorin rings in the open-sandwich dimer, and with several amino acid side chains of neighboring protein subunits.
The photoprotection of Chls in WSCP relies anyway on the isoprenoids, even in the absence of carotenoids, in the form of the phytyl chains. The recently proposed phytyl-based photoprotective mechanism of WSCP is not only dependent on the presence of the phytyl moiety but even on its correct conformation.
Amino acid exchanges in a single position in the apoprotein affect the localization of phytyls with regard to their chlorin rings, modifying the ability of phytyls to photoprotect these pigments. These findings highlight how interactions between Chl substructures and their protein environment determine the properties of Chl—protein complexes.
Materials and Methods. Mutagenesis and transformation of DNA encoding Lv-wt uniprot: O and various single mutants see Table S1 were performed as described before. In the UV region — nm , the parameters were as follows: 0. Supporting Information. Author Information. Daniel M. The authors declare no competing financial interest. Science , , — , DOI: Anbar, Ariel D.
American Association for the Advancement of Science. Correlations with org. Molybdenum and rhenium were probably supplied to Archean oceans by oxidative weathering of crustal sulfide minerals. These findings point to the presence of small amts. Crown group Oxyphotobacteria postdate the rise of oxygen. Geobiology , 15 , 19 — 29 , DOI: The rise of oxygen ca. This event was enabled by the evolution of oxygenic photosynthesis in the ancestors of Cyanobacteria.
However, long-standing questions concern the evolutionary timing of this metab. Recently, knowledge of the Cyanobacteria phylum has expanded with the discovery of non-photosynthetic members, including a closely related sister group termed Melainabacteria, with the known oxygenic phototrophs restricted to a clade recently designated Oxyphotobacteria.
By integrating genomic data from the Melainabacteria, cross-calibrated Bayesian relaxed mol. We further est. Together, these results are consistent with the hypothesis that oxygenic photosynthesis evolved relatively close in time to the rise of oxygen. Photosynthesis research , 99 2 , ISSN: Chlorophyll a Chl a serves a dual role in oxygenic photosynthesis: in light harvesting as well as in converting energy of absorbed photons to chemical energy.
They trap the long wavelength electronic excitation, and convert it into chemical energy. Chl a appears to be unique and irreplaceable, particularly if global scale oxygenic photosynthesis is considered.
Its uniqueness is determined by its physicochemical properties, but there is more. Other contributing factors include specially tailored protein environments, and functional compatibility with neighboring electron transporting cofactors. Why chlorophyll? Spectra of porphyrins. Absorption spectra for 3 series of porphyrins derived from the basic skeleton are given: a compds. The spectra are discussed in terms of a 4-orbital model. Emission spectra of metal porphyrins are discussed.
In closed shell metals, the continuous enhancement of phosphorescence at the expense of fluorescence is attributed to spin-orbit coupling. In paramagnetic metals, observed effects are attributed to the existence of a state at the same energy as the usual triplet, but with multiplicity the same as the ground state; its intensity is ascribed to exchange interactions. In diamagnetic metals with unfilled d shells, peculiar emission properties are attributed to enhanced spin orbit coupling due to low-lying metal triplets.
The Evolution of Chlorophylls and Photosynthesis. In Chlorophylls and Bacteriochlorophylls ; Grimm, B. Effects of substituents on synthetic analogs of chlorophylls. Part 2: Redox properties, optical spectra and electronic structure. Part 2: redox properties, optical spectra and electronic structure. Blackwell Publishing, Inc. Results are also given for a zinc and free base oxophorbine, which contain the keto-bearing isocyclic ring present in the natural photosynthetic pigments such as chlorophyll a.
A general finding is that the 3,13 positions are more sensitive to the effects of auxochromes than the 5,10,15 positions. In addn. The calcns. Collectively, the results and anal. These insights form a framework that underpins the rational design of chlorins for applications encompassing photomedicine and solar-energy conversion. Why are plants green-relationships between pigment absorption and photosynthetic efficiency. Photosynthetica , 10 , — Google Scholar There is no corresponding record for this reference.
Understanding chlorophylls: Central magnesium ion and phytyl as structural determinants. Acta, Bioenerg. Elsevier B. Phytol, a C20 alc. To evaluate their intramol. A detailed spectroscopic study on the model pigments reveals meaningful differences in the spectral characteristics of the phytylated and non-phytylated pigments.
Their anal. However, both residues significantly influence the conformation of the pigment macrocycle and the removal of either residue increases the macrocycle flexibility. The chelation of Mg has a flattening effect on the macrocycle whereas bulky phytyl residue seems to control the conformation of the chromophore via steric interactions with ring V and its substituents.
The anal. These observations imply that the truncated chlorophylls used in theor. Intrinsic Photoprotective Mechanisms in Chlorophylls. Photosynthetic energy conversion competes with the formation of chlorophyll triplet states and the generation of reactive oxygen species.
These may, esp. Many sophisticated photoprotective mechanisms have evolved to secure a harmless flow of excitation energy through the photosynthetic complexes. Time-resolved laser-induced optoacoustic spectroscopy was used to compare the properties of the T1 states of pheophytin a and its metallocomplexes. The lowest quantum yield of the T1 state is always obsd.
Axial coordination to the central Mg further lowers the yield of both T1 and singlet oxygen. These results reveal the existence of intrinsic photoprotective mechanisms in chlorophylls, embedded in their mol. Such intrinsic photoprotective effects must have created a large evolutionary advantage for the Mg complexes during their evolution as the principal photoactive cofactors of photosynthetic proteins.
Chlorophyll and Photosynthesis. B , , — , DOI: Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein WSCP from Virginia pepperweed Lepidium virginicum , a unique WSCP that preferentially binds chlorophyll b in vitro.
Planta , , — , DOI: Various plants possess non-photosynthetic, hydrophilic chlorophyll Chl proteins called water-sol. Chl-binding proteins WSCPs. Although the physicochem. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry anal. RT-PCR anal. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.
Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein. American Society for Biochemistry and Molecular Biology.
A water-sol. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum Virginia pepperweed to clarify the mechanism of its photoprotective function. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl mols. With ref. Biochemistry , 56 , — , DOI: Palm, Daniel M. Chlorophylls are the most abundant pigments in plants. Dark green, leafy vegetables like spinach are rich sources of natural chlorophylls.
The chlorophyll contents of selected vegetables are presented in Table 1 Green algae like chlorella are often marketed as supplemental sources of chlorophyll.
Because natural chlorophyll is not as stable as chlorophyllin and is much more expensive, most over-the-counter chlorophyll supplements actually contain chlorophyllin. Oral preparations of sodium copper chlorophyllin also called chlorophyllin copper complex are available in supplements and as an over-the-counter drug Derifil used to reduce odor from colostomies or ileostomies or to reduce fecal odor due to incontinence Sodium copper chlorophyllin may also be used as a color additive in foods, drugs, and cosmetics Natural chlorophylls are not known to be toxic, and no toxic effects have been attributed to chlorophyllin despite more than 50 years of clinical use in humans 8 , 23 , When taken orally, chlorophyllin may cause green discoloration of urine or feces, or yellow or black discoloration of the tongue There have also been occasional reports of diarrhea related to oral chlorophyllin use.
When applied topically to wounds, chlorophyllin has been reported to cause mild burning or itching in some cases Oral chlorophyllin may result in false positive results on guaiac card tests for occult blood Since the safety of chlorophyll or chlorophyllin supplements has not been tested in pregnant or lactating women, they should be avoided during pregnancy and lactation. Originally written in by: Jane Higdon, Ph.
Updated in December by: Jane Higdon, Ph. Updated in June by: Victoria J. Drake, Ph. Reviewed in June by: Roderick H. Dashwood, Ph. Sudakin DL. Dietary aflatoxin exposure and chemoprevention of cancer: a clinical review.
J Toxicol Clin Toxicol. Dashwood RH. The importance of using pure chemicals in anti mutagenicity studies: chlorophyllin as a case in point. Mutat Res. Identification and characterization of chlorin e 4 ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial. Chem Res Toxicol. Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen.
Study of the forces of stabilizing complexes between chlorophylls and heterocyclic amine mutagens. Environ Mol Mutagen. Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen.
Chemoprevention with chlorophyllin in individuals exposed to dietary aflatoxin. Scavenging of reactive oxygen species by chlorophyllin: an ESR study. Free Radic Res. Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo. Biochim Biophys Acta. Inhibitory effects of chlorophyllin, hemin and tetrakis 4-benzoic acid porphyrin on oxidative DNA damage and mouse skin inflammation induced by O-tetradecanoylphorbolacetate as a possible anti-tumor promoting mechanism.
Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo. Non-specific inhibition of cytochrome P activities by chlorophyllin in human and rat liver microsomes. Effect of dietary constituents with chemopreventive potential on adduct formation of a low dose of the heterocyclic amines PhIP and IQ and phase II hepatic enzymes.
Nutr Cancer. E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin. Int J Cancer. Chemopreventive properties of chlorophyllin: inhibition of aflatoxin B1 AFB1 -DNA binding in vivo and anti-mutagenic activity against AFB1 and two heterocyclic amines in the Salmonella mutagenicity assay.
Use of aflatoxin adducts as intermediate endpoints to assess the efficacy of chemopreventive interventions in animals and man. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Dietary chlorophyllin is a potent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout. Cancer Res. Post-initiation chlorophyllin exposure does not modulate aflatoxin-induced foci in the liver and colon of rats. J Carcinog. A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China.
Cancer Epidemiol Biomarkers Prev. Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Biological activities of chlorophyll derivatives. N J Med. Siegel LH. The control of ileostomy and colostomy odors. Weingarten M, Payson B. Deodorization of colostomies with chlorophyll. Rev Gastroenterol. Ugeskr Laeger. Use of chlorophyllin in the care of geriatric patients. J Am Geriatr Soc. Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients.
Life Sci. Kephart JC. Chlorophyll derivatives - their chemistry, commercial preparation and uses. Econ Bot. Bowers WF. Chlorophyll in wound healing and suppurative disease. Am J Surg. Carpenter EB. Clinical experiences with chlorophyll preparations.
Stamford: Thomson Health Care, Inc. Smith RG. Enzymatic debriding agents: an evaluation of the medical literature. Ostomy Wound Manage. Weir D, Farley KL. J Wound Ostomy Continence Nurs.
0コメント