The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.     

Remodeling of excitation energy transfer in extremophilic red algal PSI-LHCI complex during light adaptation

Photosynthetic PSI-LHCI complexes from an extremophilic red alga C. merolae grown under varying light regimes are characterized by decreasing size of LHCI antenna with increasing illumination intensity [1]. In this study we applied time-resolved fluorescence spectroscopy to characterize the kinetics of energy transfer processes in three types of PSI-LHCI supercomplexes isolated from the low (LL), medium (ML) and extreme high light (EHL) conditions. We show that the average rate of fluorescence decay is not correlated with the size of LHCI antenna and is twice faster in complexes isolated from ML-grown cells (~25–30 ps) than from both LL- and EHL-exposed cells (~50–55 ps). The difference is mainly due to a contribution of a long ~100-ps decay component detected only for the latter two PSI samples. We propose that the lack of this phase in ML complexes is caused by perfect coupling of this antenna to PSI core and lack of low-energy chlorophylls in LHCI. On the other hand, the presence of the slow, ~100-ps, fluorescence decay component in LL and EHL complexes may be due to the weak coupling between PSI core and LHCI antenna complex, and due to the presence of particularly low-energy or red chlorophylls in LHCI. Our study has revealed the remarkable functional flexibility of light harvesting strategies that have evolved in the extremophilic red algae in response to harsh or limiting light conditions involving accumulation of low energy chlorophylls that exert two distinct functions: as energy traps or as far-red absorbing light harvesting antenna, respectively.     

Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae

The binding affinity of the two substrate–water molecules to the water-oxidizing Mn₄CaO₅ catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S₂ and S₃ states by the exchange of bound ¹⁶O-substrate against ¹⁸O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: k_f = 52 ± 8 s^− 1 and k_s = 1.9 ± 0.3 s^− 1 in the S₂ state, and k_f = 42 ± 2 s^− 1 and k_slow = 1.2 ± 0.3 s^− 1 in S₃, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S₂ state, which confirms beyond doubt that both substrate–water molecules are already bound in the S₂ state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S₂ → S₃ transition. Implications for recent models for water-oxidation are briefly discussed.     

Non-reductive iron release from horse spleen ferritin using desferoxamine chelation

The rate of Fe³⁺ release from horse spleen ferritin (HoSF) was measured using the Fe³⁺-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k₁); 2) a second slower first-order reaction in HoSF (k₂); and 3) a zero-order slow reaction in HoSF (k₃). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6-9 rapidly reacting Fe³⁺ ions. Activation energies of 10.5 ± 0.8, 13.5 ± 2.0 and 62.4 ± 2.1 kJ/mol were calculated for the kinetic events associated with k₁, k₂, and k₃, respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k₃ and corresponding to the dissociation of Fe³⁺ ions from the FeOOH core that bind to an Fe³⁺ binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe³⁺ from site 1 to site 2 (a second binding site in the 3-fold channel) (k₂); and 3) rapid iron loss from site 2 to DES (k₁).     

Simultaneous true, gated, and coupled electron-transfer reactions and energetics of protein rearrangement

Cytochromes c₆ and f react by three et mechanisms under similar conditions. We report temperature and viscosity effects on the protein docking and kinetics of ³Zncyt c₆ + cyt f(III) → Zncyt c₆⁺ + cyt f(II). At 0.5-40.0 °C, this reaction occurs within the persistent (associated) diprotein complex with the rate constant k^pr and within the transient (collision) complex with the rate constant k^tr. The viscosity independence of k^pr, the donor-acceptor coupling H_ab = (0.5 ± 0.1) cm⁻¹, and reorganizational energy λ = (2.14 ± 0.02) eV indicate true et within the persistent complex. The viscosity dependence of k^tr and a break at 30 °C in the Eyring plot for k^tr reveal mechanisms within the transient complex that are reversibly switched by temperature change. Kramers protein friction parameters differ much for the reactions below (σ = 0.3 ± 0.1, δ = 0.85 ± 0.07) and above (σ = 4.0 ± 0.9, δ = 0.40 ± 0.06) 30 °C. The transient complex(es) undergo(es) coupled et below ca. 30 °C and gated et above ca. 30 °C. Brownian dynamics simulations reveal two broad, dynamic ensembles of configurations “bridged” by few intermediate configurations through which the interconversion presumably occurs.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k_off) is reported. In the absence of drugs, the value of k_off is (1.3 ± 0.2) × 10⁻⁴ s⁻¹. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k_off value increases to (8.6 ± 0.9) × 10⁻⁴ s⁻¹. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) × 10⁻³ M and (6.2 ± 0.7) × 10⁻⁵ M, respectively) were determined. The increase of k_off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow’s site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.     

Steady-state kinetic mechanism of LodA,a novel cysteine tryptophylquinone-dependent oxidase

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of k_cat of 0.22 ± 0.04 s⁻¹, K_lysine of 3.2 ± 0.5 μM and K_O2 of 37.2 ± 6.1 μM. The k_cat exhibited a pH optimum at 7.5 while k_cat/K_lysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O₂ in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.     

Kinetics and mechanism of the reactions of ozone with superoxo, hydroperoxo, and hydrido complexes of rhodium(III)

Oxidation of the title complexes with ozone takes place by hydrogen atom, hydride, and electron transfer mechanisms. The reaction with (NH₃)₄(H₂O)RhH²⁺ is a two electron process, believed to involve hydride transfer with a rate constant k = (2.2 ± 0.2) × 10⁵ M⁻¹ s⁻¹ and an isotope effect k_H/k_D = 2. The oxidation of (NH₃)₄(H₂O)RhOOH²⁺ to (NH₃)₄(H₂O)RhOO²⁺ by an apparent hydrogen atom transfer is quantitative and fast, k = (6.9 ± 0.3) × 10³ M⁻¹ s⁻¹, and constitutes a useful route for the preparation of the superoxo complex. The latter is also oxidized by ozone, but more slowly, k = 480 ± 50 M⁻¹ s⁻¹.     

Relationship between change in core temperature and change in cortisol and TNFα during exercise

The combined thermal load created by exercise and a hot environment is associated with an exaggerated core temperature response. The elevated core temperature is believed to increase the total stress of the exercise. Increased stress during exercise has been associated with increased levels of cortisol. The association of cortisol with increased inflammatory responses following exercise in the heat is equivocal. Thus, the purpose of the current investigation was to explore the relationship between increases in rectal temperature (T_re) and TNFα and cortisol. To induce T_re changes, 8 male subjects (mean±SD, age=23.6±2 yr, VO₂max=52.8±3.7 mL/kg/min, BMI=24.2±1.9) participated in two 40 min trials of cycle ergometry at 65% of VO₂peak immersed to chest level in cool (25 °C) and warm (38.5 °C) water. T_re was monitored throughout each trial, with blood samples taken immediately pre and post of each trial. Neither cortisol nor TNFα changed significantly during exercise in the cool water; however, in the warm trial, both cortisol and TNFα significantly increased (p<0.004). Concordance correlations (R_c) between Δ cortisol and Δ TNFα indicated a strong but non-significant correlation (R_c=0.833, p=0.135). In conclusion, changes in core temperature may be impacting the relationship between exercise induced changes in cortisol and TNFα. Therefore, acute moderate-intensity exercise (40 min or less) in warm water impacts the stress and inflammatory response. Understanding this is important because exercise load may need to be adjusted in warm and hot environments to avoid the negative effects of elevated stress and inflammation response.     

Kinetics of the cis-to-planar interconversion of cis-diaqua(1,4,7,11-tetraazacyclotetradecane)nickel(II) ion

In order to examine the effects of coordinated hydroxide ion and free hydroxide ion in configurational conversion of a tetraamine macrocyclic ligand complex, the kinetics of the cis-to-planar interconversion of cis-[Ni(isocyclam)(H₂O)₂]²⁺ (isocyclam, 1,4,7,11-tetraazacyclotetradecane) has been studied spectrophotometrically in basic aqueous solution. The interconversion requires the inversion of one sec-NH center of the folded cis-complex to have the planar species. Kinetic data are satisfactorily fitted by the rate law, R = k_OH[OH⁻][cis-[Ni(isocyclam)(H₂O)₂]²⁺], where k_OH = 3.84 × 10³ dm³ mol⁻¹ s⁻¹ at 25.0 ± 0.1 °C with I = 0.10 mol dm⁻³ (NaClO₄). The large ΔH^≠, 61.7 ± 3.2 kJ mol⁻¹, and the large positive ΔS^≠, 30.2 ± 10.8 J K⁻¹ mol⁻¹, strongly support a free-base-catalyzed mechanism for the reaction.     

Kinetic study of base-catalyzed asymmetric nitrogen conversion of cis-folded macrocyclic nickel(II) complex of 1,4,7,11-tetraazacyclotetradecane

In order to examine the effects of coordinated hydroxide ion and free hydroxide ion in configurational conversion of a tetraamine macrocyclic ligand complex, the kinetic of the cis-to-planar interconversion of cis-[Ni(isocyclam)(H₂O)₂]²⁺ (isocyclam = 1,4,7,11-tetraazacyclotetradecane) has been examined spectrophotometrically. All kinetic data have been satisfactorily fitted by the rate law, R = (k₁K_OH[OH⁻]² + k₂[OH⁻])(1 + K_OH[OH⁻])⁻¹(cis-[Ni(isocyclam)(H₂O)₂]²⁺ + [Ni(isocyclam)(OH)]⁺), where k₂ = (3.40 ± 0.12) × 10³ dm³ mol⁻¹ s⁻¹ is almost equal to k_OH determined in buffer solution (lowly basic media), K_OH = 22.7 ± 1.4 dm³ mol⁻¹ at I (ionic strength) = 0.10 mol dm⁻³ (NaClO₄ + NaOH) and 25.0 °C. Rate constants, k₂ and K_OH, are functions of ionic strength, giving a good evidence for an intermolecular pathway. The reaction follows a free-base-catalyzed mechanism where nitrogen inversion, solvation and ring conformational changes are occurred.     

Diurnal variation in relative photosynthetic performance in giant kelp Macrocystis pyrifera (Phaeophyceae, Laminariales) at different depths as estimated using PAM fluorometry

Pulse-amplitude modulated (PAM) fluorometry allows instantaneous estimates of photosynthetic rates, but may well produce variable measurements of photosynthetic activity depending on time of day, recent light history, internal fluctuations, and environmental variability. To investigate this, we compare estimates of diurnal variability in relative photosynthetic performance for the giant kelp, Macrocystis pyrifera (L.) C. Agardh, obtained from PAM fluorometry at three depths during 3 days characterized by different light conditions, and for two different blade ages. Sampling in the mid morning, late morning, early afternoon and late afternoon, we examined diurnal changes in relative photosynthetic performance in meristematic tissue and older blades occurring near the bottom, in the mid water, and at the water surface. Measures of maximum relative electron transport rates (rETR_max), minimum saturating irradiance (E_k), photosynthetic efficiency (α) and maximum quantum yield (F_v/F_m) show that giant kelp blades in the mid water and near the bottom exhibit little to no photosynthetic changes during the day. Near the surface, however, blades exhibit photosynthetic characteristics similar to light-adapted species in that they begin the day acclimated to low light, acclimate to increasing irradiance during the day, and end the day acclimated to low light. Consequently, while estimates of rETR_max were highest during the midday for all sample depths and days, they were also always highest near the surface for both old blades (112.16 ± 8.7, 98.6 ± 14.7, 70.16 ± 5.7) and meristematic tissue (109.0 ± 9.0, 86.9 ± 1.9, 59.2 ± 11.6, surface, mid water and bottom, respectively). Similar patterns were observed for E_k for both old blades (169.2 ± 5.4, 88.0 ± 11.2, 83.8 ± 5.2) and meristematic tissue (138.4 ± 11.5, 96.6 ± 4.69, 68.4 ± 10.6). In contrast, estimates of F_v/F_m were lowest near the surface during the midday for both old blades (0.6 ± 0.02, 0.73 ± 0.69, 0.75 ± 0.01) and meristematic tissue (0.58 ± 0.02, 0.69 ± 0.05, 0.74 ± 0.01, surface, mid water and bottom, respectively). These patterns coincided with similar patterns in ambient light, which was most variable and reached its greatest values near the surface during the midday.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.