Studying the structural properties of polyalanine and polyglutamine peptides

Poly-(Ala) and poly-(Gln) peptides have important biological effects, and can cause various human illnesses and neurodegenerative diseases. Conformational analysis of these homo-oligopeptides (HOPs) was carried out by simulated annealing in order to identify their structural properties regarding secondary structures and intramolecular H-bonding patterns. Poly-(Ala) and poly-(Gln) peptides composed of 7, 10, 14 or 20 amino acids were modelled in both charged and terminally blocked forms. In the case of conformers derived from simulated annealing calculations, the presence of various secondary structural elements (different types of β-turns, α-helix, 3₁₀-helix, poly-proline II helix, parallel and antiparallel β-strands) was investigated. Moreover, the intramolecular H-bonding patterns formed either between the backbone atoms for both HOPs or between the backbone and side-chain atoms for the poly-(Gln) peptides were examined. Our results showed that different secondary structural elements (type I and type III β-turns, α-helix, 3₁₀-helix, antiparallel β-strand) could be observed in both poly-(Ala) and poly-(Gln) peptides and, according to their presence, characteristic H-bonding patterns formed mainly by i←i+3 and i←i+4 H-bonds could be found.     

HIV-1 GP120 V3 conformational and informational entropies

In an attempt to analyze structure, function and evolution of HIV-1 GP120 V3, interactions among the Hartree–Fock energy, the conformational entropy and the Shannon entropy were determined for the 1NJ0 set of antibody-bound V3 loop conformers. The Hartree–Fock energy of each conformer was determined at the MINI level with GAMESS. The conformational entropy was determined per conformer and per residue from the mass-weighted covariance matrices. The Shannon entropy per residue was determined from sequence-substitution frequencies. Correlations were determined by linear regression analysis. There was a negative correlation between the Hartree–Fock energy and the conformational entropy (R=−0.4840, p=0.0078, df =28) that enhanced the negative Helmholtz-free-energy change for the binding of the GP120 ligand to target CD4. The Shannon entropy of V3 was a function of the conformational entropy variance (R=0.7225, p=0.00157, df=15) and of the V3 Hartree–Fock energy. Biological implications of this work are that (1) conformational entropy interacts with V3 Hartree–Fock energy to enhance GP120 binding to CD4 cell receptors and that (2) the Hartree–Fock energy of V3 interacts with the evolutionary system to participate in the regulation of V3 diversity.     

Studies on the interaction between benzophenone and bovine serum albumin by spectroscopic methods

The interaction between benzophenone (BP) and bovine serum albumin (BSA) was investigated by the methods of fluorescence spectroscopy combined with UV–Vis absorption and circular dichroism (CD) measurements under simulative physiological conditions. The experiment results showed that the fluorescence quenching of BSA by BP was resulted from the formation of a BP–BSA complex and the corresponding association constants (K _a) between BP and BSA at four different temperatures had been determined using the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –43.73 kJ mol⁻¹ and −53.05 J mol⁻¹ K⁻¹, respectively, which suggested that hydrogen bond and van der Waals force played major roles in stabilizing the BP–BSA complex. Site marker competitive experiments indicated that the binding of BP to BSA primarily took place in site I (sub-domain IIA). The conformational investigation showed that the presence of BP decreased the α-helical content of BSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.     

Element Concentrations in Some Species of Seaweeds from La Paz Bay and La Paz Lagoon, South-Western Baja California, Mexico

La Paz Bay and La Paz Lagoon are water bodies of the Gulf of California that are influenced by waste water discharges from the City of La Paz and from activities of the phosphorite mining company “Rofomex”. Because seaweeds concentrate elements from the water and are used as effective indicators of contamination by metals, we investigated their usefulness in this region. Concentrations of certain major elements (Ca, Fe, K and Na) and trace elements (As, Ba, Co, Cr, Cs, Hf, Rb, Sb, Sc, Se, Sr, Ta, Th, U, Zn and Zr) were determined in 12 species of seaweeds from La Paz Bay and La Paz Lagoon using instrumental neutron activation analysis. The contents of trace elements of environmental importance (As, Co, Cr, Fe, Sb, Se and Zn) in all studied samples are within the range of typical levels for a pristine environment not subjected to anthropogenic impact. Somewhat higher concentrations of Cr (81mg kg⁻¹), Hf (4mg kg⁻¹), Rb (48mg kg⁻¹), Sc (6.3mg kg⁻¹), Ta (0.95mg kg⁻¹), Th (6.8mg kg⁻¹), U (33mg kg⁻¹) and Zn (90mg kg⁻¹) were found in the green seaweed species Ulva (formerly Enteromorpha) intestinalis, whereas such elements as As (77mg kg⁻¹), Sb (1.4mg kg⁻¹) and Se (1.8mg kg⁻¹) were mainly concentrated in the species Sargassum sinicola, Codium cuneatum and Padina mexicana respectively. Because of their higher abundance and heterogeneity in elemental composition the seaweeds species Ulva intestinalis and Caulerpa sertularioides seem to be more suitable for further biomonitoring of heavy metal pollution of the coastal waters in this zone.     

Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies

In this work, fluorescence spectroscopy in combination with circular dichroism spectroscopy and molecular modeling was employed to investigate the binding of 10-hydroxycamptothecin (HCPT) to human serum albumin (HSA) under simulative physiological conditions. The experiment results showed that the fluorescence quenching of HSA by HCPT was a result of the formation of HCPT–HSA complex. The corresponding association constants (K _a) between HCPT and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The results of thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bonds and van der Waals forces played major roles for HCPT–HSA association. Site marker competitive displacement experiment indicated that the binding of HCPT to HSA primarily took place in sub-domain IIA (site I). Molecular docking study further confirmed the binding mode and the binding site obtained by fluorescence and site marker competitive experiments. The conformational investigation showed that the presence of HCPT decreased the α-helical content of HSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of HSA molecules.     

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

 Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D₂O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54  °C in horse and at 57  °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35  °C, followed by a second amide II transition at 50  °C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50  °C (tuna) and 54  °C (horse) are also observed during the thermal titration of the CN^–-ligated cytochromes (where CN^– displaces the Met80 ligand), but a well-defined 35  °C amide II transition is absent from the titration curve of the CN^–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5  °C range with T _m values at 74  °C (bovine c), 70  °C (horse c) and 65  °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25  °C. Curve fitting of the amide I (H₂O) and amide I′ (D₂O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN^– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D₂O is a powerful tool in protein conformational analysis. Received: 1 April 1999 / Accepted: 24 August 1999     

Spectroscopic Detection of Pseudo-Turns in Homodetic Cyclic Penta- and Hexapeptides Comprising β-Homoproline

β-Amino acids with side chains at C² and/or at C³ are of growing interest in drug design, as they may induce astonishing and unusual peptide conformations. Therefore it is of eminent importance to gather information on the consequences of β-amino acid incorporation on the three-dimensional structure of a peptide. This paper describes the synthesis and conformational analysis of cyclic penta- and hexapeptides comprising either (S)-Pro or (S)-β-Hpro. The conformational influence of the β-homoproline building block was analyzed by the combined application of CD, FT-IR and NMR. While the CD spectra of the proline containing peptides indicate the presence of inverse γ-turns and βII-turns, the CD spectra of the β-homoamino acid analogs are dominated by an unprecedented negative band near 205 nm associated with a pseudo-β-turn (Ψβ) or pseudo-γ-turn (Ψγ). These results were confirmed by FT-IR spectroscopy, which also indicates the formation of two internal hydrogen bonds in the cyclic peptides containing the β-homoproline. The conformations of the β-homoproline containing pentapeptides were additionally determined by NMR in combination with MD simulations in two different solvents. The conformation in trifluoroethanol (TFE) is characterized by a bifurcated hydrogen bond stabilizing a pseudo-γ-turn with β-homoproline in the central position, nested with a pseudo-β-turn with β-homoproline in the i+1 position. The combined CD/FT-IR studies clearly show that the replacement of proline by β-homoproline gives rise to a more flexible peptide backbone, and CD spectroscopy hints towards the presence of pseudo-β- or pseudo-γ-turns.     

Relationships between molecular states (conformation and orientation) and activities of α-amylase adsorbed on ultrafine silica particles

Bacillus subtilis α-amylase, which contains a relatively large amount of α-helix, was adsorbed on two types of ultrafine silica particles (silica-1 and-2, average diameter 15 nm) under various conditions. The changes in circular dichroism (CD) spectra of α-amylase upon adsorption were measured, and the extent of conformational changes was estimated from the reduction in α-helix content. In additions the activities of adsorbed α-amylase were measured at pH 5.2 using corn starch andp-nitrophenylbenzyl α-maltopentaoside (BG5P). In the ultrafine silica-2 particles, the extent of both activity reductions and conformational changes upon adsorption was much larger than that in the ultrafine silica-1 particles and increased with decreasing pH and amount of adsorption. The extent of activity reductions correlated closely with the conformational changes. On the other hand, the effect of reduction in α-amylase activity upon adsorption measured by BG5P was smaller than that measured by starch, indicating that the lack of accessibility of the active site to a large substrate also reduces the activity of adsorbed α-amylase. However, the effects of particle type and adsorption conditions on the extent of activity reductions by the accessibility resistance were small. Therefore, variation of the activity of adsorbed α-amylase is mainly attributable to the extent of conformational changes upon adsorption. Based on these results, a procedure to prepare adsorbed α-amylase with high activity was investigated.     

Epitope mapping from real time kinetic studies — Role of cross-linked disulphides and incidental interacting regions in affinity measurements: Study with human chorionic gonadotropin and monoclonal antibodies

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (α5-14 and α55-62). The association rate constant (k₊₁) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope-paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region α35-47 and α60-84. The IIRs contribute significantly towards theK _A of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb,K _A is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.     

Ultrastructure and elemental composition of the eggshell of Reeve’s pheasant ( Syrmaticus reevesii )

The eggshell of Reeve’s pheasant (Syrmaticus reevesii) collected from the Dongzhai National Nature Reserve in Henan Province, China was studied. By using scanning electron microscopy and inductively coupled plasma (ICP) spectrometry, the ultrastructure and elemental composition of the eggshell was determined. The study showed that the average thickness of the surface layer of crystals, the palisade and cone layer, and the eggshell membrane were 20.8, 220.8 and 62.5 μm, respectively, accounting for 6.8%, 72.6% and 20.6% of the total thickness of the eggshell. There were many vesicular holes in the palisade layer with an average diameter of 0.32 ± 0.08 μm (n = 30). The function of these holes might be significant to air exchange. The shape of the eggshell pore on the surface layer of crystals is round or elliptical. The fracture surface of the pore is funnel-shaped. Some granules filled the upper part of the eggshell pores. The content of 21 elements in the eggshell of wild and captive Reeve’s pheasants was compared and presented. It indicated that among the elements that made up the eggshell of the wild pheasant, the content of Ca, Mg, P and S was much higher, ω > 1 mg/g, with ω (Ca) being higher than 40% of the eggshell. The contents of Na, Si, Sr, K and Al were ω = 0.1–1 mg/g, while Fe, Zn, Pb, Mn, Cu, V and Ti had lower concentrations (ω = 1–100 μg/g). The ω of Ni, Cr, Co, Se, Cd were lower than 1 μg/g. The elemental composition in the eggshell of the captive Reeve’s pheasant kept in the Dongzhai National Natural Reserve was significantly different from that of the wild species, with a difference of over 20% on S, Cu, Fe, Al, Mn, Si, Sr, Se and Cr. The lower intake of Fe, Mn, Si and Sr on the one hand and the higher intake of S, Cu, Al and Cr on the other hand might be responsible for the low fertility of captive Reeve’s pheasants in the Dongzhai National Nature Reserve. In order to ensure that the pheasants are receiving the proper amount of nutrition and to improve their breeding success, the amount of certain elements in the food should be adjusted. Translated from Journal of Beijing Normal University (Natural Science), 2006, 42(1): 78–82 [译自: 北京师范大学学报 (自然科学版)]     

Trophic Transfer and Dietary Mineral Intake of Essential Elements in <Emphasis Type="Italic">Thunnus albacares</Emphasis> and <Emphasis Type="Italic">Katsuwonus pelamis</Emphasis> from the Eastern Pacific

With the aim of knowing levels of Cu, Fe, and Zn in the edible portion of tunas Thunnus albacares and Katsuwonus pelamis, these elements were measured in 73 fish collected in the eastern Pacific. Additionally, trophic transfer (TF) and dietary mineral intake (DMI) of analyzed metals were determined. The most elevated average concentration of Zn (31.7 μg g⁻¹ dry weight) was found in T. albacares from the Baja California region; in the case of Cu and Fe, highest values (3.86 and 71.0 μg g⁻¹ dry weight) were found in K. pelamis from the offshore waters in the Pacific Ocean. Concentrations of Cu in muscle tissue of both species were significantly (p < 0.05) correlated (negatively) with total length of specimens; levels of Fe in muscle of K. pelamis also showed a negative correlation with total length. None of TFs were above the unit, it implies that there is no biomagnification of Cu, Fe, and Zn. The DMI of Cu, Fe, and Zn from muscle tissue of analyzed tuna represented less than 13%, 21%, and 8%, respectively, of the daily requirements for humans.     

A molecular mechanics and semiempirical molecular orbital study on the conformation of polynorbornene chains

The conformational analysis of polynorbornene (PNB) chains was investigated with the AM1, MM2, AMBER and OPLS methods taking into consideration the possibility of binding of norbornene monomers to each other at various positions, i.e. exo–exo, exo–endo, endo–endo. The chain that is formed by connecting exo–endo positions of the monomers has lower torsional barrier energy than those formed with bonds at other positions and has more flexibility. It is determined that the thredisyndiotactic chain formed by exo–endo addition adopts a helix structure and has a coil shape. The disyndiotactic chain formed by connecting norbornene monomers in mixed type has a linear structure. It is found that the repeat unit conformations of thredisyndiotactic and disyndiotactic chains of PNB are TGTG^– and (TGTG^–)₂, respectively.     

Conformational Effects of a Common Codon 399 Polymorphism on the BRCT1 Domain of the XRCC1 Protein

The X-ray cross-complementing-1 (XRCC1) protein functions as a scaffold that coordinates the activity of the cellular machinery involved in base excision repair (BER) of DNA damage. The BRCT1 domain of XRCC1 is responsible for interacting with several of the key components of the BER machinery, and it is also the site of a common genetic polymorphism in XRCC1 at amino acid residue 399 (Arg → Gln). Experimental and epidemiologic evidence suggest that this polymorphism may alter BER capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XRCC1 induced by the polymorphism. Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the BRCT1 domain of XRCC1, and differences in structure produced by the polymorphic substitution were determined. The results indicate that, although the general configuration of both proteins is similar and there is little actual deviation at the site of the polymorphism itself, the substitution produces significant conformational changes at several other sites in the BRCT1 domain, including the loss of secondary structural features such as α helices that may be critical for protein–protein interactions. These results provide support for the hypothesis that this polymorphism in XRCC1 could affect DNA repair capability by altering the structure of the BRCT1 domain and thus the ability of XRCC1 to coordinate BER.     

Identification of IS elements in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains grown in a medium with ferrous iron or adapted to elemental sulfur

IS elements were identified in the genomes of five Acidithiobacillus ferrooxidans strains isolated from various media. IST2 elements were revealed in all the strains grown in a medium with ferrous iron, ISAfe1 elements were detected in four strains (TFBk, TFL-2, TFV-1 and TFO). Three strains (TFV-1, TFN-d and TFO) were found to contain IS elements, ~600 bp long. These were named preliminary as ISAfe600. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of an ISAfe1 element in TFBk and TFL-2 strains and complete sequencing of the ISAfe1 element in the TFBk strain has revealed nucleotide substitutions as compared to the prototype, i.e., the ISAfe1 element of an ATCC 19859 strain. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of the IST2 elements in TFO, TFBk and TFL-2 strains has shown numerous nucleotide substitutions when compared to the IST2 element of an ATCC 19859 strain. Complete sequencing of the IST2 element in the TFBk strain has revealed: the divergence between the IST2 elements in the TFBk strain and the prototype was 21.2%. Southern hybridization of EcoRI fragments of the chromosomal DNA from five A. ferrooxidans strains grown in a medium with ferrous iron using an internal region of ISAfe1, a full-length ISAfe1 or a full-length IST2 as probes has shown them to differ in the number of copies of IS elements and their localization on the chromosomes. Adaptation to elemental sulfur in A. ferrooxidans strains caused changes in the number, intensity and localization of hybridization bands. The authors discuss the role of IS elements in the adaptation of A. ferrooxidans to the new energy substrate. The nucleotide sequence data reported in this paper were deposited in GenBank under accession numbers: AY823401, the ISAfe1 from A. ferrooxidans TFBk; AY825254, the IST2 from TFBk; DQ002894, the 5′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ002895, the 3′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ005952, the 5′-terminal nucleotide sequence of IST2 from TFV-1; DQ005953, the 3′-terminal nucleotide sequence of IST2 from TFV-1.     

Performance Traits and Metabolic Responses in Goats (<Emphasis Type="Italic">Capra hircus)</Emphasis> Supplemented with Inorganic Trivalent Chromium

The effects of supplemental chromium (Cr) as chromic chloride hexahydrate in incremental dose levels (0, 0.5, 1.0, and 1.5 mg/day for 240 days) on metabolism of nutrients and trace elements were determined in dwarf Bengal goats (Capra hircus, castrated males, average age 3 months, n = 24, initial mean body weight 6.4 ± 0.22 kg). Live weight increased linearly (p < 0.05) with the level of supplemental Cr. Organic matter and crude protein digestibility, intake of total digestible nutrients, and retention of N (g/g N intake) increased (p < 0.05) in a dose-dependent linear manner. Serum cholesterol and tryacylglycerol concentrations changed inversely with the dose of supplemental Cr (p < 0.01). Supplemental Cr positively influenced retention of copper and iron (p < 0.05) causing linear increase (p < 0.01) in their serum concentrations. It was concluded that Cr supplementation may improve utilization of nutrients including the trace elements and may also elicit a hypolidemic effect in goats. However, further study with regards to optimization of dose is warranted.     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.     

Extremely Low-Frequency Magnetic Field Decreased Calcium,Zinc and Magnesium Levels in Costa of Rat

Electromagnetic field (EMF) can affect cells due to biochemical change followed by a change in level of ions trafficking through membrane. We aimed to investigate possible changes in some elements in costa of rats exposed to long-term extremely low-frequency magnetic field (ELF-MF). Rats were exposed to 100 and 500 μT ELF-MF, which are the safety standards of public and occupational exposure for 2 h/day during 10 months. At the end of the exposure period, the samples of costa were taken from the rats exposed to ELF-MF and sham. The levels of elements were measured by using atomic absorption spectrophotometry (AAS) and ultraviolet (UV) spectrophotometry. Ca levels decreased in the ELF-500 exposure group in comparison to sham group (p < 0.05). Statistically significant decrease was found in Mg levels in the ELF-500 exposure group in comparison to sham and ELF-100 exposure groups (p < 0.05). Zn levels were found to be lower in the ELF-500 exposure group than those in the sham and ELF-100 exposure groups (p < 0.05). No significant differences were determined between groups in terms of the levels of P, Cu and Fe. In conclusion, it can be maintained that long-term ELF-MF exposure can affect the chemical structure and metabolism of bone by changing the levels of some important elements such as Ca, Zn and Mg in rats.     

The charge-transfer complex between protochlorophyllide and NADPH: an intermediate in protochlorophyllide photoreduction

A hypothesis describing the mechanism of photoactive protochlorophyllide (P) photoreduction in vivo, relating mainly to the molecular nature of the intermediates, is proposed. The hypothesis is compatible with currently published experimental data. After illumination of etiolated barley leaves at 143 to 153 K, the absorption of P remains essentially unchanged, but a new absorption band at 690 nm is observed. Appearance of this new intermediate enables to distinguish between light and dark stages of the photoconversion reaction. When returned to the higher temperature in the dark, the treated leaves begin accumulating chlorophyllide (Chlide), concomitant with the disappearance of the 690-nm band. The decay time of the excited P (P^*) is estimated at 300 ps, which approximates the time constant of photoinduced electron transfer (ET). It is suggested that the charge-transfer complex (CTC) in its ground state (GS) (ground state of CTC formed by the partial (δ) electron transfer), i.e. (P^δ−•••H–D^δ+), between P and NADPH – the electron and proton donor (H–D) – accumulates in the following sequence: P^* + H–D → (P^*•••H–D)→[(P^*•••H–D)←(P⁻•••H–D⁺)] → ¹(P⁻•••H–D⁺)] → ³(P⁻•••H–D⁺) → (P^δ−•••H–D ^δ+), where an equilibrium state (ES) – [(P^*•••H–D)←(P⁻•••H–D⁺)] – with a lifetime of about 1 to 2 ns, exists between the local excited (LE) and ET states. The existence of a triplet ET state – ³(P⁻•••H–D⁺) – is proposed because the time interval between recording of the ES and appearance of the CTC GS (35–250 ns) does not fit the lifetime of the singlet excited complex (exciplex). It is feasible that apart from NADPH, other intermediate proton carriers are contemporaneously involved in the dark reaction (P^δ−•••H–D^δ+) → Chlide, because proton binding to the C₇–C₈ bond in vivo takes place in the trans-configuration. The hydride ion may approach the C₇–C₈ bond from one side by heterolytic fission and an additional proton, donated by the protein group, may be simultaneously added to this bond from the opposite side of the porphyrin nucleus surface. This revised version was published online in June 2006 with corrections to the Cover Date.