Time-resolved UV-visible Absorption Spectroscopy Research Articles

Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51.

Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV-visible absorption spectroscopy. In both enzymes, the P450 → P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 → P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme-thiolate ligand.

Free-Radical-Mediated Photoinduced Electron Transfer between 6-Thioguanine and Tryptophan Leading to DNA-Protein-Like Cross-Link.

The nucleobase analog 6-thioguanine (6-TG) has emerged as important immunosuppressant, anti-inflammatory, and anticancer drug in the past few decades, but its unique photosensitivity of absorbing strongly ultraviolet UVA light elicits photochemical hazards in many ways. The particularly intriguing yet unresolved question is whether the direct photoreaction of 6-TG can promote DNA-protein cross-links (DPCs) formation, which are large DNA adducts blocking DNA replication and physically impede DNA-related processes. Herein, by real-time observation of radical intermediates using time-resolved UV-vis absorption spectroscopy in conjunction with product analysis by HPLC-MS, we discover that UVA excitation of 6-TG triggers direct covalent cross-linking with tryptophan (TrpH) via an exquisite radical mechanism of electron transfer. The photoexcitation prepares the redox-active triplet 36-TG*, which initiates electron transfer with TrpH, creating TrpH•+ and 6-TG•- in the first step. The deprotonated Trp• undergoes radical-recombination with its geminate partner 6-TG•- and eliminates a H2S, leading to the cross-linking product 6-TG-Trp. The photoadduct structures (two chiral isomers and one constitutional isomer) are identified unambiguously, validating further the mechanism. These findings pinpoint the exact amino acid that is vulnerable to photo-cross-linking with 6-TG and establish a mechanistic framework for understanding mutagenic DPCs formation and developing photoprobes based on this new type of photo-cross-linking.

Insights into enzymatic mimicking activity of silver nanoprisms: spectral monitoring and analysis

Peroxidase-like reaction process involving o-phenylenediamine (OPD) and silver nanoprisms in the presence of hydrogen peroxide (H2O2) was monitored using time-resolved ultraviolet–visible (UV–Vis) absorption spectroscopy. The oxidation of OPD and etching of silver nanoprisms were investigated by analyzing the dynamic spectral data. Two-dimensional correlation spectroscopy (2D-COS) and principal component analysis (PCA) were employed to gain insights into the correlation between catalytic oxidation of OPD and etching of silver nanoprisms. It was found that OPD offered significant protection effect for silver nanoprisms so that morphologies of silver nanoprisms maintained at the beginning period after addition of H2O2. Moreover, silver nanoprisms accelerated the oxidation of OPD by H2O2, demonstrating enzymatic mimicking activity of silver nanoprisms. The combination of time-resolved UV–Vis absorption spectroscopy and spectral calculation methods could be used for exploration of complex reaction systems with spectral variations.

Probing the role of arginine 323 of the D1 protein in photosystem II function.

The Mn4 CaO5 cluster of photosystem II (PSII) advances sequentially through five oxidation states (S0 to S4 ). Under the enzyme cycle, two water molecules are oxidized, O2 is generated and four protons are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn4 CaO5 cluster and TyrZ via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site-directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S-state-dependent stoichiometry of the proton release were affected. However, several differences have been found: (1) the P680 + decay in the hundreds of ns time domain was much slower in the mutant, (2) the S2 QA - /DCMU and S3 QA - /DCMU radiative charge recombination occurred at higher temperatures and (3) the S0 TyrZ • , S1 TyrZ • , S2 TyrZ • split EPR signals induced at 4.2 K by visible light from the S0 TyrZ , S1 TyrZ , S2 TyrZ , respectively, and the (S2 TyrZ • )' induced by NIR illumination at 4.2 K of the S3 TyrZ state differed. It is proposed that the R323 residue of the D1 protein interacts with TyrZ likely via the H-bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H-bonds around TyrZ by interacting with the bulk, thus tuning the driving force required for TyrZ oxidation.

Chemical control of competing electron transfer pathways in iron tetracyano-polypyridyl photosensitizers.

Photoinduced intramolecular electron transfer dynamics following metal-to-ligand charge-transfer (MLCT) excitation of [Fe(CN)4(2,2′-bipyridine)]2− (1), [Fe(CN)4(2,3-bis(2-pyridyl)pyrazine)]2− (2) and [Fe(CN)4(2,2′-bipyrimidine)]2− (3) were investigated in various solvents with static and time-resolved UV-Visible absorption spectroscopy and Fe 2p3d resonant inelastic X-ray scattering (RIXS). This series of polypyridyl ligands, combined with the strong solvatochromism of the complexes, enables the 1MLCT vertical energy to be varied from 1.64 eV to 2.64 eV and the 3MLCT lifetime to range from 180 fs to 67 ps. The 3MLCT lifetimes in 1 and 2 decrease exponentially as the MLCT energy increases, consistent with electron transfer to the lowest energy triplet metal-centred (3MC) excited state, as established by the Tanabe–Sugano analysis of the Fe 2p3d RIXS data. In contrast, the 3MLCT lifetime in 3 changes non-monotonically with MLCT energy, exhibiting a maximum. This qualitatively distinct behaviour results from a competing 3MLCT → ground state (GS) electron transfer pathway that exhibits energy gap law behaviour. The 3MLCT → GS pathway involves nuclear tunnelling for the high-frequency polypyridyl breathing mode (hν = 1530 cm−1), which is most displaced for complex 3, making this pathway significantly more efficient. Our study demonstrates that the excited state relaxation mechanism of Fe polypyridyl photosensitizers can be readily tuned by ligand and solvent environment. Furthermore, our study reveals that extending charge transfer lifetimes requires control of the relative energies of the 3MLCT and the 3MC states and suppression of the intramolecular distortion of the acceptor ligand in the 3MLCT excited state.

PH-Dependent Morphology and Photoresponse of Azopyridine-Terminated Poly(N-isopropylacrylamide) Nanoparticles in Water.

A series of azopyridine-terminated poly(N-isopropylacrylamide)s (PNIPAM) (C12-PN-AzPy) (∼5000 < Mw < 20 000 g mol–1, polydispersity index 1.25 or less) were prepared by reversible addition–fragmentation chain-transfer polymerization of NIPAM in the presence of a chain-transfer agent that contains an AzPy group and an n-dodecyl chain. In cold water, the polymers form nanoparticles (5.9 nm < Rh < 10.9 nm) that were characterized by light scattering (LS), 1H NMR diffusion experiments, and high-resolution transmission electron microscopy. We monitored the pH-dependent photoisomerization of C12-PN-AzPy nanoparticles by steady-state and time-resolved UV–vis absorption spectroscopy. Azopyridine is known to undergo a very fast cis-to-trans thermal relaxation when the azopyridine nitrogen is quaternized or bound to a hydrogen bond donor. The cis-to-trans thermal relaxation of the AzPy chromophore in an acidic nanoparticle suspension is very fast with a half-life τ = 2.3 ms at pH 3.0. It slows down slightly for nanoparticles in neutral water (τ = 0.96 s, pH 7.0), and it is very slow for AzPy-PNIPAM particles in alkaline medium (τ > 3600 s, pH 10). The pH-dependent dynamics of the cis-to-trans dark relaxation, supported by Fourier transform infrared spectroscopy, 1H NMR spectroscopy, and LS analysis, suggest that in acidic medium, the nanoparticles consist of a core of assembled C12 chains surrounded by a shell of hydrated PNIPAM chains with the AzPy+ end groups preferentially located near the particle/water interface. In neutral medium, the shell surrounding the core contains AzPy groups H-bonded to the amide hydrogen of the PNIPAM chain repeat units. At pH 10.0, the amide hydrogen binds preferentially to the hydroxide anions. The AzPy groups reside preferentially in the vicinity of the C12 core of the nanoparticles. The morphology of the nanoparticles results from the competition between the segregation of the hydrophobic and hydrophilic components and weak attractive interactions, such as H-bonds between the AzPy groups and the amide hydrogen of the PNIPAM repeat units.

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), S = 1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), S = 5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZ → S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZ → S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZ → S2HSTyrZ intermediate step before the S2HSTyrZ → S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZ → S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S = 3 S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.

Binding Interactions of Zinc Cationic Porphyrin with Duplex DNA: From B-DNA to Z-DNA.

Recognition of unusual left-handed Z-DNA by specific binding of small molecules is crucial for understanding biological functions in which this particular structure participates. Recent investigations indicate that zinc cationic porphyrin (ZnTMPyP4) is promising as a probe for recognizing Z-DNA due to its characteristic chiroptical properties upon binding with Z-DNA. However, binding mechanisms of the ZnTMPyP4/Z-DNA complex remain unclear. By employing time-resolved UV-visible absorption spectroscopy in conjunction with induced circular dichroism (ICD), UV-vis, and fluorescence measurements, we examined the binding interactions of ZnTMPyP4 towards B-DNA and Z-DNA. For the ZnTMPyP4/Z-DNA complex, two coexisting binding modes were identified as the electrostatic interaction between pyridyl groups and phosphate backbones, and the major groove binding by zinc(II) coordinating with the exposed guanine N7. The respective contribution of each mode is assessed, allowing a complete scenario of binding modes revealed for the ZnTMPyP4/Z-DNA. These interaction modes are quite different from those (intercalation and partial intercalation modes) for the ZnTMPyP4/B-DNA complex, thereby resulting in explicit differentiation between B-DNA and Z-DNA. Additionally, the binding interactions of planar TMPyP4 to DNA were also investigated as a comparison. It is shown that without available virtual orbitals to coordinate, TMPyP4 binds with Z-DNA solely in the intercalation mode, as with B-DNA, and the intercalation results in a structural transition from Z-DNA to B-ZNA. These results provide mechanistic insights for understanding ZnTMPyP4 as a probe of recognizing Z-DNA and afford a possible strategy for designing new porphyrin derivatives with available virtual orbitals for the discrimination of B-DNA and Z-DNA.

The low spin - high spin equilibrium in the S2-state of the water oxidizing enzyme

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2LS and S2HS is pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the S2LS is formed first and advances to S3via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS to S3 transition to occur below 200 K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.

Fast kinetics studies of the Lewis acid-base complexation of transient stannylenes with σ- and π-donors in solution.

The Lewis acid-base complexation chemistry of dimethyl- and diphenylstannylene (SnMe2 and SnPh2, respectively) in hexanes solution has been studied by laser photolysis methods. Complexation of the two stannylenes with a series of nine O-, S-, and N-donors (including cyclic and acyclic dialkyl ethers and sulfides, a primary, secondary, and tertiary amine, ethyl acetate and acetone), two alkenes, and an alkyne proceeds rapidly and reversibly to generate the corresponding stannylene-donor Lewis pairs, which have been detected in each case by time-resolved UV-vis absorption spectroscopy. The complexes exhibit UV-vis absorption maxima in the range of λmax ∼ 310-405 nm depending on the donor and substitution at tin. Bimolecular rate constants for complexation (kC), which could be determined for 14 of the 24 Lewis pairs that were studied, were found to fall within a factor of four of the diffusional limit in all cases, with SnMe2 showing consistently higher reactivity than SnPh2. Equilibrium constants for complexation (KC) could be measured for all but one of the stannylene-π- and O-donor pairs, the values corresponding to (gas phase) binding free energies in the range of +1.1 to -3.9 kcal mol-1. Comparison of the experimental equilibrium constants for complexation of SnMe2 and SnPh2 with methanol and diethyl ether to those measured previously for the homologous silylenes and germylenes indicates that Lewis acidity decreases in the order SiR2 > SnR2 > GeR2 for both the dimethyl- and diphenyltetrylene series, the diphenyl derivatives exhibiting significantly stronger Lewis acidity in all three cases. The experimental trends in the binding energies and UV-vis spectra of the complexes are reproduced well by density functional theory (DFT) calculations, which have been carried out at the (TD)ωB97XD/def2-TZVP level of theory. The experimental data also show evidence of a reaction between tetramethyldistannene (Me2Sn[double bond, length as m-dash]SnMe2, 4a) and amine donors, which is suggested to afford the corresponding amine-stabilized stannylidenestannylene structure. The mechanistic proposal is supported by DFT calculations of the complexation of 4a and SnMe2 with model O-, S- and N-donors.

Kinetico-mechanistic studies on methemoglobin generation by biologically active thiosemicarbazone iron(III) complexes

The oxidation of human oxyhemoglobin (HbO2) to methemoglobin (metHb) is an undesirable side effect identified in some promising thiosemicarbazone anti-cancer drugs. This is attributable to oxidation reactions driven by FeIII complexes of these drugs formed in vivo. In this work the FeIII complexes of selected 2-benzoylpyridine thiosemicarbazones (HBpT), 2-acetylpyridine thiosemicarbazones (HApT), and the clinically trialled thiosemicarbazone, Triapine® (3-amino-2-pyridinecarboxaldehyde thiosemicarbazone, H3-AP), have been studied. This was achieved by time-resolved UV–Visible absorption spectroscopy and the sequential oxidation of the α- and β-chains of HbO2 at distinctly different rates has been identified. A key structural element, namely a terminal –NH2 group on the thiosemicarbazone moiety, was found to be an important common feature of the most active HbO2 oxidising complexes that were investigated. Therefore, these studies indicate that an unsubstituted –NH2 moiety at the terminus of the thiosemicarbazone group should be avoided in the design of future compounds from this class.

P75: H2S induced S-nitrosoglutathione decomposition – Effect of thiols, ph and oxygen

Hydrogen sulfide (H 2 S) and nitric oxide (NO) have some similar biological properties. Interaction of NO with sulfhydryl group of thiols leads to the formation of Snitrosothiols, the biological reservoirs of NO. Snitrosoglutathione (GSNO) is the most abundant and relatively stable S-nitrosothiol. We studied the effect of low molecular thiols, pH and oxygen on the reaction of H 2 S donor Na 2 S (0.2 mM) with GSNO (0.2 mM) by time resolved UV–vis absorption spectroscopy. We found that this decomposition is pH dependent, the maximum rate is at physiological pH 7.4, higher or lower pH decelerated the reaction (7.4 > 8.0 ≫ 6.0). Next we studied the effect of low molecular thiols - L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH) on this reaction. At physiological and alkaline pH (7.4 and 8.0), they had a deceleration effect (NAC > GSH > Cys). At acidic pH (6.0), they had the reverse effect – acceleration of GSNO decomposition (Cys > GSH > NAC). Oxidized thiols (oxidized glutathione or cystine) had no effect at any studied pH values. Measurements in nitrogen deaerated solutions revealed that oxygen is not necessary for H 2 S induced GSNO decomposition, although it slightly accelerates the reaction. Presented results can contribute to understanding of the involvement of H 2 S and low molecular thiols in NO signaling pathways. This work was supported by VEGA 2/0050/13 and APVV-0074-11.

Comparison between Spontaneous and Photoinduced Ionization Mechanisms for p-Quaterphenyl in M-ZSM-5 (M = H+, Na+) Zeolites

The electron transfers following the initial ionization of a probe molecule (p-quaterphenyl) adsorbed in the channels of M-ZSM-5 (M = H+, Na+) zeolites are investigated using various complementary spectroscopic techniques. Under the same reaction conditions, ionization occurs spontaneously during molecule diffusion in the acid H-ZSM-5 whereas charge separation needs to be photoinduced within the pores of Na-ZSM-5. The electron transfer processes are found to be identical for both the cases in terms of transient species formed before the final charge recombination. The initial ionization leads to the formation of a radical cation which evolves gradually toward an electron/hole pair associated with a charge transfer complex. However, the reaction kinetics depend dramatically on the ionization way. As spontaneous ionization is associated with the sorption process, it is closely correlated with the molecule diffusion and, thus, is very slow due to the bulky size of the molecule. In this case, radical cations and subsequent charge transfer complexes are stabilized for months in high yield and are clearly characterized by diffuse reflectance UV–vis absorption, EPR, and Raman scattering in resonance and in off-resonance conditions. After photoionization, the evolution is followed as a function of time by using time-resolved UV–vis absorption spectroscopy on a large scale of time extending from a few microseconds to several days.

Structural, magnetic and electronic characterization of an isostructural series of dinuclear complexes of 3d metal ions bridged by tpbd

Dinuclear complexes of the type [{M(H2O)(phen)}2(μ-tpbd)](ClO4)4 with N,N,N′,N′-Tetrakis-(2-pyridylmethyl)-benzene-1,4-diamine (tpbd) as bridging ligand and M=Mn(II) (1), Fe(II) (2), Co(II) (3), Ni(II) (4), Cu(II) (5), Zn(II) (6) were synthesized, structurally analyzed and their magnetic as well as their electrochemical properties are determined. A ligand-centered one-electron oxidation leads to radical complexes for which the lifetimes are strongly dependent on the coordinated metal ion as followed from time-resolved UV–Vis absorption spectroscopy. In addition to the six homometallic dinuclear complexes, one analog heterometallic Mn(II)/Ni(II) compound (7) of the same constitution was structurally and magnetically investigated, confirming an high spin ground state.

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5 cluster and TyrZ, a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn4CaO5 cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I− for Cl− in the cyanobacterium Thermosynechococcus elongatus with either Ca2+ or Sr2+ biosynthetically assembled into the Mn4 cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl−/I− exchange considerably slowed down the two S3TyrZ•→(S3TyrZ•)′→S0 reactions in which the fast phase, S3TyrZ•→(S3TyrZ•)′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on TyrZ• and the slow phase corresponds to the S0 and O2 formations and to a second proton release. The t1/2 for S0 formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the TyrZ• reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca2+ binding site and the Cl− binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl− and Ca2+ binding sites by which proton release may be channelled.

Reductive Reaction Mechanisms of the Azo Dye Orange II in Aqueous Solution and in Cellulose: From Radical Intermediates to Products

Reductive reaction mechanisms of the azo dye Orange II (Acid Orange 7) in aqueous solution have been studied from radical intermediates through to the final products using a combination of nanosecond time-resolved UV-visible absorption spectroscopy, steady-state photolysis, and HPLC techniques. The dye is reduced by photochemically produced 2-hydroxy-2-propyl radicals at a near-diffusion-controlled rate (k2 = 2.2 x 10(9) dm3 mol(-1) s(-1)) to give the dye radical anion, which then disproportionates (k3 = 2.6 x 10(8) dm3 mol(-1) s(-1)) to re-form the parent dye and a hydrazine. The hydrazine decomposes to 4-aminobenzenesulfonate and a naphthylimine species, which hydrolyses to give 1,2-naphthoquinone; this naphthoquinone and 4-aminobenzenesulfonate react to give a species that reacts further in the presence of air to form an indophenol dye. The reduction of Orange II has also been studied in cellophane, where the rate constant for dye reduction by 2-hydroxy-2-propyl radicals is approximately two orders of magnitude lower than that in aqueous solution.

Spectroscopic and Kinetic Study of the Gas-Phase CH3I−Cl and C2H5I−Cl Adducts

Time-resolved UV-visible absorption spectroscopy has been coupled with UV laser flash photolysis of Cl2/RI/N2/X mixtures (R = CH3 or C2H5; X = O2, NO, or NO2) to generate the RI-Cl radical adducts in the gas phase and study the spectroscopy and reaction kinetics of these species. Both adducts were found to absorb strongly over the wavelength range 310-500 nm. The spectra were very similar in wavelength dependence with lambda(max) approximately 315 nm for both adducts and sigma(max) = (3.5 +/- 1.2) x 10(-17) and (2.7 +/- 1.0) x 10(-17) cm(2) molecule(-1) (base e) for CH3I-Cl and C2H5I-Cl, respectively (uncertainties are estimates of accuracy at the 95% confidence level). Two weaker bands with lambda max approximately 350 and 420 nm were also observed. Over the wavelength range 405-500 nm, where adduct spectra are reported both in the literature and in this study, the absorption cross sections obtained in this study are a factor of approximately 4 lower than those reported previously [Enami et al. J. Phys. Chem. A 2005, 109, 1587 and 6066]. Reactions of RI-Cl with O2 were not observed, and our data suggest that upper limit rate coefficients for these reactions at 250 K are 1.0 x 10(-17) cm(3) molecule(-1) s(-1) for R = CH3 and 2.5 x 10(-17) cm(3) molecule(-1) s(-1) for R = C2H5. Their lack of reactivity with O2 suggests that RI-Cl adducts are unlikely to play a significant role in atmospheric chemistry. Possible reactions of RI-Cl with RI could not be confirmed or ruled out, although our data suggest that upper limit rate coefficients for these reactions at 250 K are 3 x 10(-13) cm(3) molecule(-1) s(-1) for R = CH3 and 5 x 10(-13) cm(3) molecule(-1) s(-1) for R = C2H5. Rate coefficients for CH3I-Cl reactions with CH3I-Cl (k9), NO (k22), and NO2 (k24), and C2H5I-Cl reactions with C2H5I-Cl (k14), NO (k23), and NO2 (k25) were measured at 250 K. In units of 10(-11) cm(3) molecule(-1) s(-1), the rate coefficients were found to be 2k9 = 35 +/- 12, k22 = 1.8 +/- 0.4, k24 = 3.3 +/- 0.6, 2k14 = 40 +/- 16, k23 = 1.8 +/- 0.3, and k25 = 4.0 +/- 0.9, where the uncertainties are estimates of accuracy at the 95% confidence level.

Biosynthetic Exchange of Bromide for Chloride and Strontium for Calcium in the Photosystem II Oxygen-evolving Enzymes

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.

Spectroscopic studies of the I 2/O 3 photochemistry : Part 2. Improved spectra of iodine oxides and analysis of the IO absorption spectrum

Based on multivariate statistical separation techniques spectra of iodine oxides formed in the photolysis of I 2 + O 3 have been obtained. Data was recorded by time resolved UV–vis absorption spectroscopy. Overlapped spectra of ground state IO( ν′ ← 0), vibrationally excited IO( ν′ ← ν″) with ν″ > 0, and OIO have been separated from each other and from underlying absorptions of further iodine oxides. The uncertainty due to uncorrected foreign absorptions is of no more than ±3%. Relative errors of spectra are of the order of a few percent for the main parts of the spectra. The up to now unknown continuous parts of spectra are thereby determined. Previous uncertainties in differential absolute absorption cross-section are removed. By the separation of the ground state IO spectrum from that of vibrationally excited IO spectroscopic measurements under non-equilibrium conditions are enabled. Three further absorber spectra have been extracted in the 200–600 nm window. Two of them are most likely caused by I 2O 2 and possibly I 2O 3 thereby providing part of the missing link between IO and OIO consumption and the formation of higher oxides and possibly aerosol. All spectra are available as supplementary data. Given the unprecedented quality of extracted spectra for the first time an analysis of band strength of the IO(A 2Π 3/2 ← X 2Π 3/2) transition could be made. The continuum absorption of the ground state IO spectrum was resolved into overlapped bands of bound–bound transitions and two bound-free transitions. From the bound-free transitions found the existence of two optically active repulsive states intersecting with the IO(A 2Π 3/2) potential has been inferred and tangents to the two repulsive potentials have been determined. Correlating the tangents to probable dissociation products gives a plausible rough picture of the shape of the repulsive states which is in good agreement with previous observations on predissociation of states. There is evidence that one of the continuous absorptions observed could have its origin in the IO( 2Π 1/2) sub-system. Relative band strength was determined for absorption bands of IO(A 2Π 3/2 ← X 2Π 3/2). They are in good agreement with literature and with simple calculations based on a Morse approximation. An anomalous behaviour of IO(2 ← 0) in time resolved measurements was observed and studied. It could be explained by a hypothetical partial population inversion of the IO(A 2Π 3/2) state, but the source of it remains unclear.

Spectroscopic and kinetic study of the gas-phase CS 2[sbnd]Cl adduct

Time-resolved UV–vis absorption spectroscopy (TRUVVAS) has been coupled with 248 nm laser flash photolysis (LFP) of Cl 2CO in the presence of CS 2 (and in some cases O 2, NO or NO 2) to generate the CS 2 Cl radical adduct in the gas phase and study the spectroscopy and kinetics of this species. CS 2 Cl is found to possess a strong absorption band at λ max ≈ 365 nm with σ max = (2.3 ± 0.7) × 10 −17 cm 2 molecule −1 (base e) and a weaker band at λ max ≈ 480 nm; the gas-phase spectrum of CS 2 Cl is very similar to the previously reported liquid-phase spectrum in CCl 4 solvent. Reaction of CS 2 Cl with O 2 is found to be very slow; our data suggest that the rate coefficient for this reaction at 240 K is less than 5.0 × 10 −18 cm 3 molecule −1 s −1. Rate coefficients for CS 2 Cl reactions with CS 2 Cl ( k 3), NO ( k 5), and NO 2 ( k 6) were measured at 240 K and, in units of 10 −11 cm 3 molecule −1 s −1, were found to be 2 k 3 = 15 ± 6, k 5 = 2.2 ± 0.5, and k 6 = 1.3 ± 0.4, where the uncertainties are estimates of accuracy at the 95% confidence level.