Raman spectroscopic analysis of the sodium salt of kappa-carrageenan and related compounds in solution

Laser-Raman spectra of Na⁺ kappa-carrageenan, Na⁺ neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm⁻¹ are reported for solutions in H₂O and D₂O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm⁻¹, close to the maximum of the symmetrical COS stretching (∼850 cm⁻¹). The symmetrical SO stretch is proposed to occur near 1040 cm⁻¹ and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm⁻¹. The intense band in the region 730–740 cm⁻¹ is ascribed to a complex ring vibration.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77–84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with |D| = 279·10⁻⁴ cm⁻¹ and |E| = 39·10⁻⁴ cm⁻¹. The triplet resonances titrate with a midpoint potential of +380 ± 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and ΔH_pp = 1.0 mT. Deconvolution of the P-700⁺ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species, A₀, which is characterized by g = 2.0033 ± 0.0004 and ΔH_pp = 1.0 ± 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A₁ in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A₁ is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A₁ in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A₁ from its binding site.     

Preparation and characterization of oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) complexes of 3,3′-(1,2-phenylenediimino)diacrolein

The oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) of 3,3′-(1,2-phenylenediimino)diacrolein were prepared and investigated by means of mass, electronic, vibrational, NMR and ESR spectroscopy as well as magnetic susceptibility measurements. The acetatomanganese(III) and chloroiron(III) complexes were confirmed to be of high spin type. The absorption bands appearing in the energy range greater than 23 000 cm⁻¹ were attributed to π→π^* transitions within a ligand molecule and charge- transfer transitions from metal to ligand. The metal complexes assume the square-planar configuration type since the ligand-field bands were detected in the 12 700–18 500 cm⁻¹ region. Strong bands appearing at 1601 and 1627 cm⁻¹ were assigned to the CC and CO stretching vibrational modes, respectively, and were shifted to lower frequency upon metal-coordination. A VO stretching band was observed at 982 cm⁻¹ for the oxovanadium(IV) complex and a CO stretching band was observed at 1547 cm⁻¹ for the acetatomanganese(III) complex. Upon complex formation the amine proton signal is found to vanish and the aldehydic methine proton signal in the lowest field is shifted upfield for the nickel(II), zinc(II) and palladium(II) complexes. ¹³C NMR spectra support the coordination structure of the complexes which is revealed by ¹H NMR spectra. As judged by the spin Hamiltonian parameters, the oxovanadium(IV) complex is of a square- planar type with an unpaired electron in the d_xy orbital and the copper(II) complex assumes a distorted square-planar coordination due to the presence of five- and six-membered chelate rings with an unpaired electron in the d_x2−y2 orbital.     

Quantitative IR spectroscopic determination of the concentrations of cobalt carbonyl complexes in a three-component system

The three-component system consisting of Co₄(CO)₁₂, Co₂(CO)₈ and HCo(CO)₄ was analyzed by means of IR spectroscopy. A quantitative method was developed in order to enable the precise calculation of the concentrations of all three compounds simultaneously. The quantitative analysis was based upon the intensity of the bridging CO stretching bands at 1858 cm⁻¹ (A₂) and 1867 cm⁻¹ (A₁) of the polynuclear carbonyls, and the terminal CO symmetric stretching band of HCo(CO)₄ at 2116 cm⁻¹ (A₃). The mathematical expression for the concentrations of the three compounds required the precise knowledge of at least one of the four extinction coefficients of either Co₄(CO)₁₂, ϵ_J1 and ϵ_J2 or Co₂(CO)₈, ϵ_K1 and ϵ_K2. The reference extinction coefficient was ϵ_K2, because Co₂(CO)₈ was employed as the starting compound in all experiments performed in this study. In order to determine the extinction coefficient of HCo(CO)₄ at 2116 cm⁻¹, ϵ₃, intensities of this band were plotted as function of the corresponding concentrations of HCo(CO)₄, which were calculated by means of the three- component system method; from the slope of the straight line ϵ₃ could be directly calculated.     

Time-resolved FTIR difference spectroscopy for the study of quinones in the A1 binding site in photosystem I: Identification of neutral state quinone bands

Infrared absorption bands associated with the neutral state of quinones in the A₁ binding site in photosystem I (PSI) have been difficult to identify in the past. This problem is addressed here, where time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study PSI with six different quinones incorporated into the A₁ binding site. (P700⁺A₁⁻ – P700A₁) and (A₁⁻ – A₁) FTIR difference spectra (DS) were obtained for PSI with the different quinones incorporated, and several double-difference spectra (DDS) were constructed from the DS. From analysis of the DS and DDS, in combination with density functional theory based vibrational frequency calculations of the quinones, the neutral state bands of the incorporated quinones are identified and assigned. For neutral PhQ in the A₁ binding site, infrared absorption bands were identified near 1665 and 1635 cm⁻¹, that are due to the C₁O and C₄O stretching vibrations of the incorporated PhQ, respectively. These assignments indicate a 30 cm⁻¹ separation between the C₁O and C₄O modes, considerably less than the ~80 cm⁻¹ found for similar modes of PhQ⁻. The C₄O mode downshifts due to hydrogen bonding, so the suggestion is that hydrogen bonding is weaker for the neutral state compared to the anion state, indicating radical-induced proton dynamics associated with the quinone in the A₁ binding site in PSI.     

FT-IR spectroscopic evidence of sugar ring conformational changes in GpC and CpG on platination and intercalation

An FT-IR spectroscopic study concerning changes in the conformation of sugar in the dinucleotides; GpC and CpG, on platination and intercalation is presented. The results are compared with the FT-IR spectral data of 5′-CMP, 5′-GMP, 3′-GMP and their metal adducts. The spectra of free GpC, free CpG, proflavine-GpC, proflavine-CpG, and cis-[Pt(NH₃)₂(GpC)₂]²⁺ exhibit the diagnostic band at 800 cm⁻¹ which was assigned to a sugar phosphate vibrational mode and diagnostic of C3′-endo sugar pucker. In the case of 9-aminoacridine-GpC and cis-[Pt(NH₃)₂(CpG]⁺ the diagnostic bands of the C2′-endo and C3′-endo conformations are observed at 810–820 cm⁻¹ and near 800 cm⁻¹ respectively. The results are in good agreement with X-ray data. The infrared diagnostic bands are important for distinguishing the sugar pucker conformational changes.     

On transverse ordering in biomembranes by Raman spectroscopy

Computer simulation analysis of the structure sensitive features in the Raman spectrum of aqueous dipalmitoyl phosphatidylcholine (DPPC) multilayers was made for the 1000–1200 cm₋₁ region. The composite triplet was resolved into 4 Lorentzian bands with the possibility to follow their parameters (amplitude, width and position) in the temperature interval 25–60°C. The analysis reveals the apparent inward shift of the 1130 and 1065 cm⁻¹ bands towards both sides of the broad intense 1087 cm⁻¹ feature which is due to the relative changes in amplitudes of these bands in the course of the lipid phase transition. The more accurate method for the evaluation of the trans order parameter (S_trans) confirms the underestimation of the trans segment content above the phase transition temperature when using both the relative amplitude and area of the 1130 cm⁻¹ band for the quantitative characterization of chain conformation. Calculated changes in the bilayer thickness following the frequency shift of the 1100 cm⁻¹ band are in good agreement with the previously reported deuterium NMR data and X-ray diffraction studies.     

Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy

Age-mediated deacetylation of hyaluronic acid and dermatan sulfate, and shift of sulfate ester configuration were indicated by infrared spectroscopy. Hyaluronic acid and the three dermatan sulfates (DS₁₈, DS₁₈ and DS₃₅), sequentially precipitated from adult skin with 18%, 28% and 35% ethanol, were analyzed at varying ages. At age 75 years, loss of infrared bands in the 1650-1600 cm⁻¹ region, at 1380 cm⁻¹ and 1320 cm⁻¹ and appearance of a band at 1560 cm⁻¹ were characteristic of hyaluronic acid and DS₃₅,·moreover, in DS₂₈ and DS₃₅ the intensities of the bands at 840 cm⁻¹ and 860 cm⁻ were, respectively, decreased and increased. A low intensity band in the 805-785 cm⁻¹ region was observed in the spectra of DS₁₈ (19–35 years), DS₂₈ (70–80 years) and DS₃₅ (all ages). It intensified in DS₂₈ of the 80-years-olds. In the 75±5-year-old group. ninhydrin-positive material of hyaluronic acid and DS₃₅ increased, while reducing GlcNAc of hyaluronic acid decreased. The data demonstrated hyaluronic acid and DS₃₅ deacetylation and suggested a decrease of equatorial sulfates with infrared band at 840 cm⁻¹ and an incrase of axial sulfates with band at 860 cm⁻¹ in DS₂₈ and DS₃₅ of the 75±5-yearl-old set. Equatorial sulfates with band in the 805±785 cm⁻¹ region apparently decreased in DS₁₈ after 35 years and increased in DS₂₈ of the oldest group.     

Photosystem I charge separation in the absence of center A and B. III. Biochemical characterization of a reaction center particle containing P-700 and FX

The Photosystem I reaction center is a membrane-bound, multiprotein complex containing a primary electron donor (P-700), a primary electron acceptor (A₀), an intermediate electron acceptor (A₁) and three membrane-bound iron-sulfur centers (F_X, F_B, and F_A). We reported in part I of this series (Golbeck, J.H. and Cornelius, J.M. (1986) Biochim. Biophys. Acta 849, 16–24) that in the presence of 1% lithium dodecyl sulfate (LDS), the reaction center becomes dissociated, resulting in charge separation and recombination between P-700 and F_X without the need for prereduction of F_A and F_B. In this paper, we report (i) the LDS-induced onset of the 1.2-ms ‘fast’ phase of the P-700 absorption transient is time-dependent, attaining a maximum 3:1 ratio of ‘fast’ to ‘slow’ kinetic phases; (ii) the ‘fast’ kinetic phase, corresponding to the P-700⁺ F_X⁻ backreaction, is stabilized indefinitely by dilution of the LDS-treated particle followed by ultrafiltration over a YM-100 membrane; (iii) without stabilization, the P-700⁺ F_X⁻ reaction deteriorates, leading to the rise of the long-lived P-700 triplet formed from the P-700⁺A_O⁻ backreaction; (iv) the ‘slow’ kinetic phase correlates with the redox and ESR properties of F_A and/or F_B, which indicates that in a minority of particles the terminal iron-sulfur protein remains attached to the reaction center core; (v) the ultrafiltered reaction center is severely deficient in all of the low molecular-weight polypeptides, particularly the 19-kDa, 18-kDa and 12-kDa polypeptides relative to the 64-kDa polypeptide(s); (vi) the stabilized particle contains 5.8 mol labile sulfide per mol photoactive P-700, reflecting largely the iron-sulfur content of F_x, but also residual F_A and F_B, on the reaction center; and (vii) the apoproteins of F_A and F_B are physically removed from the reaction center particle as indicated by the presence of protein-bound zero-valence sulfur in the YM-100 filtrate. These results are interpreted in terms of a model for Photosystem I in which F_A and F_B are located on a low-molecular-weight polypeptide and F_X is depicted as a [2Fe-2S] cluster shared between the two high-molecular-weight polypeptides Photosystem I-A1 and Photosystem I-A2.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Spectroscopic studies of pig kidney diamine oxidase-anion complexes

Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm⁻¹) and 575 nm (17 400 cm⁻¹). Anion binding produces characteristic changes in the CD spectra. N₃⁻/SCN⁻ → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm⁻¹) and 365 nm (27 400 cm⁻¹), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm⁻¹) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

EPR,ligand field spectra and antimicrobial activity of dimeric aryloxyacetatocopper(II) complexes with antipyrine

Copper(lI) complexes with aryloxyacetato ligands and antipyrine (Apy) of the general formula [Cu₂- (RCOO)₄(Apy)₂] were prepared with RCOO⁻ as phenoxyacetato, 3-methylphenoxyacetato, 4-methylphenoxyacetato, 4-chlorophenoxyacetato, 2-methyl- 4-chlorophenoxyacetato, 3-methyl-4-chlorophenoxyacetato and 2-naphthoxyacetato anions. The parameters of the triplet spin state EPR spectra at X and Q band frequencies (D|hc = 0.37 cm⁻¹, g⊥ = 2.07, g6 = 2.38, A6|c = 0.007 cm⁻¹) provide evidence of a dimeric carboxylato-bridged structure with apically bound antipyrine. The almost constant EPR data as well as the energies of the ligand field transitions at 77 K [d_x²−y² ← d_xz, d_yz (14000 cm⁻¹), d_x²−y² ← d_xy (11200 cm⁻¹) and d_x²−y² ← d_z² (9500 cm⁻¹)] indicate that the square-pyramidal geometry of the CuO₅ polyhedra is nearly unaffected by the substituents on the aryloxyacetato group. The complexes show antimicrobial activity, being most efficient against Candida albicans and Bacillus subtilis.     

5-HT receptors on identified Lymnaea neurones in culture. Pharmacological characterization of 5-HT1A receptors

The ability of the selective 5-HT_1A receptor agonist R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) to bind with 5-HT receptor(s) on cultured, identified neurones in Lymnaea stagnalis was examined. The identified neurones studied were from the buccal ganglia and the serotonin-containing cerebral giant cells (CGCs). 5-HT and its agonists were applied from puffer pipettes, whilst 5-HT antagonists were applied in the bathing medium. At 10⁻³ M, the 5-HT_1A agonist, always produced paroxysmal depolarizing shifts (PDS) while at a lower concentration (10⁻⁴ M), it always mimicked the effects of 10⁻³ M 5-HT. 8-OH-DPAT (10⁻⁴ M) and 5-HT 10⁻³ M produced dose-dependent increases in the responses they evoked. At 10⁻⁴ M, the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide hydrochloride (m-CPBG), failed to hyperpolarize most of the neurones hyperpolarized by 5-HT. At 10⁻⁴ M, the antagonists ketanserin (5-HT₂), MDL 72222 (5-HT₃), and pindobind-5-HT_1A (5-HT_1A) consistently abolished spike generation ii spontaneously active neurones. Both ketanserin and MDL 72222 failed to block the actions of 8-OH-DPAT and only partially blocked those of 5-HT, but pindobind-5-HT_1A completely, but reversibly,blocked the 8-OH-DPAT effects while greatly reducing those of 5-HT. These results suggest that 5-HT_1A receptor subtypes might be involved in the hyperpolarizing responses of the CGCs and their follower motor neurones in the buccal ganglia of Lymnaea stagnalis to 5-HT. The presence of 5-HT_1A receptors on these neurones can be considered to correspond with those found in mammals because their pharmacological responses resemble those of mammalian 5-HT_1A receptors.     

Preparation and spectroscopic properties of cobalt(III), nickel(II), copper(II) and zinc(II) complexes with a novel saturated macrocyclic ligand: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22-docosahydrodibenzo-[b,i][1,4,8,11]tetraazacyclotetradecine

1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, 20,21,22-Docosahydrodibenzo[b,i] [1,4,8,11] tetraazacyclotetradecine was prepared by hydrogenation of the benzo-analogue. Five isomers are feasible as a result of this hydrogenation but only two have been isolated: isomer A (melting point 158.5– 161.0 °C) and isomer B (melting point 194.5– 196.0 °C). The ¹³C NMR study was initiated to clear up the conformational differences between isomers. The cobalt(III), nickel(II), copper(II) and zinc(II) complexes of isomers A and B were prepared and investigated by near-ultraviolet, visible, infrared, NMR and ESR measurements. The ligand-field band in the 15 000-30 000 cm⁻¹ region for the cobalt(III), nickel(II) and copper(II) complexes provided information on their geometry around the central metal atom. That is to say, the cobalt(III) complexes are subjected to the octahedral ligand-field with axial elongation. The copper(II) complexes and the nickel- (II) complex of isomer A are subjected to the square- planar ligand-field in these complexes. The ligand- field bands for the nickel(II)complex of isomer B display the square-planar-distorted octahedral equilibrium in the coordinating solvent. ESR measurements for the copper(II) complexes also presented the spin Hamiltonian parameters in accord with the square- planar coordination. A strong band appearing at ca. 3200 cm⁻¹ was assigned to the N-H stretching mode and this band was slightly shifted to lower frequency upon metal coordination. The vibrational spectra and the conductance data provided evidences for the formation of the complexes with perchlorate ion as the counter ion. ¹³C NMR suggest that the complexes of isomer A are the cis-syn-cis form and the complexes of isomer B are the cis-anti-cis form.     

Infrared spectroscopic studies of aqueous solutions of dioxouranium(VI) and its hydrolysed products and of in situ electro-generated dioxouranium(V)

Aqueous solutions of dioxouranium(VI) (pH range 0 to 4) give rise to bands at 954 and 938 cm⁻¹ attributable to the v₃(MO₂) stretching modes of the UO₂²⁺ and (UO₂)₂(OH)_2²⁺ cations, respectively. A shoulder at 916 cm⁻¹ is assigned to the v₃(MO₂) mode of hydrolysed dioxouranium(VI) species of higher nuclearity. Infrared spectro-electrochemical studies using a thin-layer reflection-absorption cell have facilitated the study of the reduction of aqueous solutions of dioxouranium(VI) to yield dioxouranium(V) which may be further reduced to uranium(IV). The electrogeneration of dioxouranium(V) is monitored by following the increase in intensity of a band at 914 cm⁻¹ which is present in the spectra at potentials between −0.2 and −0.8 V. The dioxouranium(V) species is predominantly in the form UO₂⁺, which may be in solution or incorporated into an insoluble phase of uranium oxides which deposit onto the working electrode. The U^VO bond length is estimated to be 1.76 Å, 0.03 Å longer than the U^VIO bond in aqueous solution. The maximum concentration of UO₂⁺able to be achieved is highly dependent on the pH and is optimum at pH 3.4. Changes in the pH of the solution under study can be monitored by infrared spectroscopy during the course of the reduction by determining the relative concentrations of hydrolysed dioxouranium(VI) species.     

Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center ‘X’ and correlation with optical signal ‘A2’

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700⁺ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center ‘X’, characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700⁺ A⁻₂ reaction pair and the ESR P-700⁺ center X⁻ reaction pair to indicate that signal A₂ and center X represent the same iron-sulfur center in Photosystem I.     

Primary charge separation in native and plant pheophytin a-modified reaction centers of Chloroflexus aurantiacus: Ultrafast transient absorption measurements at low temperature

Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the B_A binding site were monitored as indicators of the formation and decay of the P⁺B_A⁻ state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P^⁎ excited state, which decay by distinct means, forming the state P⁺H_A⁻ (H_A is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P^⁎ decays in ~2 ps with the formation of P⁺H_A⁻ via a short-lived P⁺B_A⁻ intermediate in a two-step ET process P^⁎ → P⁺B_A⁻→ P⁺H_A⁻. In another population (~35 %), P^⁎ decays in ~20 ps to form P⁺H_A⁻ via a superexchange mechanism without producing measurable amounts of P⁺B_A⁻. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the H_A binding site also showed the presence of two P^⁎ populations (~2 and ~20 ps), with P^⁎ decaying through P⁺B_A⁻ only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P^⁎ → P⁺H_A⁻ superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.