Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Phosphorescence of gadolinium(III) chelates under ambient conditions

The gadolinium(III) chelates Gd(dtpaH₂), Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃ with dtpa=1,1,4,7,7-diethylenetriaminepentaacetate, hfac=hexafluoroacetylacetonate, tta=thenoyltrifluoroacetonate and qu=8-quinolinolate (or oxinate) show a phosphorescence under ambient conditions. While the UV emission of Gd(dtpaH₂) at λ_max=312 nm comes from a metal-centered ff state, the bluish (λ_max=462 nm), green (λ_max=505 nm) and red (λ_max=650 nm) luminescence of Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃, respectively, originates from the lowest-energy intraligand triplets.     

Photoredox reactivity of copper(II)-3,5-diisopropylsalicylate induced by ligand-to-metal charge transfer excitation of the copper-phenolate chromophore

The electronic spectrum of Cu^II(dps)₂ in CH₃CN with dps=3,5-diisopropylsalicylate shows a ligand field absorption at λ_max=711 nm (ε=140 M⁻¹ cm⁻¹), and a phenolate to Cu(II) ligand-to-metal charge transfer (LMCT) band at λ_max=428 nm (ε=950). LMCT excitation of Cu^II(dps)₂ leads to the reduction of Cu(II) to Cu(I). Copper(II) disappears with φ=2.8×10⁻³ at λ_irr=436 nm.     

The redox potentials of the b-type cytochromes of higher plant chloroplasts

1. In fresh chloroplasts, three b-type cytochromes exist. These are b-559_HP (λ_max, 559 nm; E_m at pH 7, +370 mV; pH-independent E_m), b-559_LP (λ_max, 559 nm; E_m at pH 7, +20 mV; pH-independent E_m) and b-563 (λ_max, 563 nm; E_m at pH 7, ?110 mV; pH-independent E_m). b-559_HP may be converted to a lower potential form (λ_max, 559 nm; E_m at pH 7, +110 mV; pH-independent E_m).2. In catalytically active b-f particle preparations, three cytochromes exist. These are cytochrome f (λ_max, 554 nm; E_m at pH 7, +375 mV, pK on oxidised cytochrome at pH 9), b-563 (λ_max, 563 nm; E_m at pH 7, ?90 mV, small pH-dependence of E_m) and a b-559 species (λ_max, 559 nm, E_m at pH 7, +85 mV; pH-independent E_m).3. A positive method of demonstration and estimation of b-559_LP in fresh chloroplasts is described which involves the use of menadiol as a selective reductant of b-559_LP.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Investigation into the mechanism of λmax shifts and their dependence on pH for the 2-hydrazinopyridine derivatives of two copper amine oxidases

Copper amine oxidases (CuAO), from Escherichia coli (ECAO) and pea seedling (PSAO) were reacted with an excess of the hydrazine derivative 2-hydrazinopyridine (2HP) to form an initial, strongly absorbing adduct, (adduct 1; λ_max 420–430 nm) formed by the covalent binding of 2HP with the active site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). Thermal incubation of buffered solutions of adduct 1 (pH 5.65–10.7) or addition of KOH solution (giving a final pH of 13–15) led isosbestically to a dramatic λ_max shift yielding adduct 2 (λ_max 520–530 nm). For both ECAO and PSAO, an increase in pH resulted in increased formation of adduct 2 with concomitant loss of adduct 1. Maximum adduct 2 formation occurred at pH 9.84 in ECAO and at pH 10.7 in PSAO. Beyond these pH levels, adduct 2 formation occurred to a much lesser extent which was independent of pH, suggesting enzyme denaturation. It is proposed that the conversion of adduct 1 to adduct 2 occurs as a result of hydrazone to azo conversion mediated by loss of a single proton, possibly to the active site base. It is further postulated that adduct formation and subsequent deprotonation can be likened to the substrate and product Schiff base complexes in the reductive half cycle of copper/TPQ containing amine oxidases. As part of this study an extinction coefficient at 280 nm was determined for ECAO by gravimetric analysis. This yielded a value of 2.1×10⁵ M⁻¹ cm⁻¹ giving rise to the need of a correction factor when estimating the protein concentration from an absorbance reading at 280 nm. Using the estimated molecular mass of 160 kDa for the homodimeric ECAO, a correction factor of 0.76 must be applied.     

Thiolysis of O-2,4-dinitrophenyltyrosines. Spectrophotometric monitoring of the reaction and its use in peptide synthesis.

The thiolytic cleavage of O-2,4-dinitrophenyl (Dnp) derivatives of phenols was applied to the synthesis of tyrosine-containing peptides. This paper describes the preparation and properties of starting materials for such syntheses and illustrates their use in the synthesis of some peptides containing tyrosine at either the C- or N-terminus. A spectrophotometric method for following the thiolytic removal of Dnp groups from O-Dnp-tyrosines was developed and used to establish optimal conditions for quantitative deblockage in aqueous and nonaqueous solvents. The method is based on the fact that upon thiolysis, the colorless solution of O-Dnp-tyrosine (λ_max at 298 nm, pH 8.5) becomes yellow due to the formation of a dinitrophenylated thiol (for S-Dnp-2-mercaptoethanol, λmax at 340 nm, pH 8.5). This gives rise to a difference spectrum with a maximum at 354 nm (Δ?_M = + 8680 M^?1 cm^?1), a minimum at 298 nm (Δ?_M = ?5900 M^?1 cm^?1) and a crossover point at 318 nm, which is different (in the 290–320 nm range) from the difference spectrum obtained upon thiolysis of N^Im-Dnp-histidine. This method provides a useful analytical tool in peptide and polypeptide synthesis as well as in protein chemistry.     

Isolation and characterization of the transient,luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction

Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reaction can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2°C and decays spontaneously to give H₂O and luciferase-bound FMN with an activation enthalpy of about 120 kJ/mol. It has an absorption spectrum (λ_max = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (λ_max = 485 nm) that matches the bioluminescence emission closely.     

Characterization of two siderophores produced by Bacillus megaterium: A preliminary investigation into their potential as therapeutic agents

BackgroundMicroorganisms produce siderophores in order to scavenge iron from the environment and this study focuses on the characterization of the two siderophores secreted by Bacillus megaterium. The general biological properties and pharmacokinetics following oral application of these compounds are reported.MethodsUnder optimized culture conditions, the siderophores were harvested, purified by chromatography and identified using LC-MS and NMR. Two dihydroxamate siderophores were isolated, schizokinen (MW = 420) and schizokinen imide (MW = 402).ResultsBoth compounds demonstrate strong antioxidant activity and were found to be relatively nontoxic to both human hepatocellular carcinoma (Huh7) and peripheral blood mononuclear cells. The siderophores possess a strong affinity for iron(III) and decrease the levels of the labile iron pool (LIP) in iron-loaded cells in a concentration-dependent manner. Schizokinen, was detected as both the free siderophore and the iron complex in the plasma and urine of rats after oral gavage.ConclusionsHowever, the bioavailability was low and thus schizokinen, like deferoxamine, has no potential as an orally active iron chelator for the treatment of systemic iron overload.General significanceBy virtue of the high affinity of schizokinen for tribasic metals, this siderophore does have considerable potential for the chelation of gallium(III) and the development of clinical diagnostic reagents.     

Active Transport of Iron in Bacillus megaterium: Role of Secondary Hydroxamic Acids

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, ⁵⁹Fe³⁺ uptake from ⁵⁹FeCl₃ or the ferric hydroxamate Desferal-⁵⁹Fe³⁺ was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from ⁵⁹FeCl₃ was markedly reduced; the two ferric hydroxamates Desferal-⁵⁹Fe³⁺ or schizokinen-⁵⁹Fe³⁺ increased both total ⁵⁹Fe³⁺ uptake and the ⁵⁹Fe³⁺ appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl₃ suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from ⁵⁹FeCl₃. Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe³⁺ as an iron source, and it was unable to incorporate ⁵⁹Fe³⁺ from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe³⁺ or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe³⁺, while retaining its capacity to utilize schizokinen-Fe³⁺.     

Is the purple color of bacteriorhodopsin maintained by lipid-protein interactions?

When bacteriorhodopsin is delipidated and purified in detergents, its purple chromophore can be reversibly titrated to a red one. The pK_a of this equilibrium depends on the nature of the detergent in which bacteriorhodopsin is dispersed. In the absence of solvating amphiphiles, lipid-free detergent-free bacteriorhodopsin is red (λ_max = 480 nm) at pH higher than 3.5.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.     

Oxidative dehydrogenation of an iron-tetraazacyclotetradecatriene complex

The oxidative dehydrogenation of the bis(N- methyl imidazole)(meso-5,5,7,12,12,14-hexamethyl- 1 4,8 11-tetraazacyclotetradeca-1,3,8-triene)iron(II) complex, forming the corresponding 1 3,8,10-tetraene product was investigated by cyclic voltammetry, spectroelectrochemistry and stopped-flow kinetics with [Fe(CN)₆]³⁻, at 25 °C, I = 0.50 M and pH 7–10. The results led to a mechanism consistent with a reversible one electron transfer process generating iron(III) species which lose a proton (pK_a = 9.77) and undergo induced electron transfer in the presence of the hexacyanoferrate(III) ion (k = 4.2 × 10⁵ M⁻¹ s⁻¹). The intermediate precursor complex (λ_max = 665 nm) formed at this step, converted to the tetraene product according to a first order kinetics, with k = 0.12 s⁻¹.     

A circular dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions.

A Circular Dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions fully confirms the occurence of a very weak Cotton effect around 435 nm in the Circular Dichroic spectrum of the reduced coenzymes and therefore the very faint transition (λ_max = 435 nm; ?_max ～ 1 M^?1 cm^?1) from which the Cotton effect originates.     

Alr0397 is an outer membrane transporter for the siderophore schizokinen in Anabaena sp. strain PCC 7120

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b₅₀ appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b_?90 was observed.4. Difference spectra attributed to (BChl)₂, Q^?_II, c type cytochrome and cytochrome b₅₀ were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromophoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin.The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (?195°C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichroism can be induced by irradiation of rhodopsin extract at ?195°C with polarized light or natural light. At photosteady state the linear dichroism disappeared.Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([θ]_max = 80 800 degrees at 335 nm, and [θ]_max = 42 600 degrees at 500 nm) at ?195°C, whereas, bathorhodopsin displayed a positive peak ([θ]_max = 43 100 degrees at 334 nm) and a negative peak ([θ]_max = 163 000 degrees at 540 nm).The change of the positive sign to negative one at α-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11—12 double bond (‘photoisomerization model’).