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In the early history of life, RNA might have had many catalytic functions as ribozymes that do not exist today. To explore this possibility, catalytically active RNAs can be identified by in vitro selection experiments. Some of these experiments are best performed in nanodroplets to prevent diffusion between individual RNA sequences. In order to explore the suitability for the large-scale in emulsio selection of water-in-oil emulsions made by passing a mixture of mineral oil, the emulsifier ABIL-EM90, and a few percent of an aqueous phase through a microfluidizer, we used dynamic light scattering to characterize the size of aqueous droplets dispersed throughout the oil. We found that seven or more passes through the microfluidizer at 8000 psi with close to half molar inorganic salts and 10% polyethylene glycol produced droplets with sizes below 100 nm that were ideal for our purposes. We also identified conditions that would produce larger or smaller droplets, and we demonstrate that the emulsions are stable over weeks and months, which is desirable for different types of in vitro selection experiments.

Description, An RNA that catalyzes the formation of GTP was developed with an emulsion-based in vitro selection system., Synthesis of RNA in early life forms required chemically activated nucleotides, perhaps in the same form of nucleoside 5′-triphosphates (NTPs) as in the contemporary biosphere. We show the development of a catalytic RNA (ribozyme) that generates the nucleoside triphosphate guanosine 5′-triphosphate (GTP) from the nucleoside guanosine and the prebiotically plausible cyclic trimetaphosphate. Ribozymes were selected from 1.6 × 1014 different randomized sequences by metabolically coupling 6-thio GTP synthesis to primer extension by an RNA polymerase ribozyme within 1016 emulsion droplets. Several functional RNAs were identified, one of which was characterized in more detail. Under optimized reaction conditions, this ribozyme produced GTP at a rate 18,000-fold higher than the uncatalyzed rate, with a turnover of 1.7-fold, and supported the incorporation of GTP into RNA oligomers in tandem with an RNA polymerase ribozyme. These results are discussed in the context of early life forms.

The dimensionless equilibrium constant for the allosteric conformation change, KΔC = 0.02602 (Knowles & Magde, linked ms 2) following binding of O2 by α-chains in Tstate Hb4/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from Tstate to Rstate, of 24.3 kJ/mol; (ii) exothermic conversion of Tstate TαO2-chains to Rstate RαO2-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the Tstate conformation is converted to the Rstate conformation. ΔGo(R(Hb4), BPG) describes the endothermic decomposition of the binary complex, THb4/BPG into RHb4 and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. 248, 2774-2777). ΔGo for the equilibrium constant for ΔGO(KΔC) and Σ ΔGo for binding of O2 by the pair of equivalent Tstate α-chains, ΔGO(Tα*O2), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O2 equilibrium binding data to the Perutz-Adair equation. ΔGo for reaction of a pair of equivalent Rstate α-chains with O2, ΔGO(RαO2), was estimated from the known affinity of myoglobin for O2 at 37oC (Theorell H. (1936). Biochem. Z., 268, 73-81), -63.4 kJ/mol. The unknown quantity, ∆GO(R(HbO2)4/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (R(HbO2)4/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to Tstate conformations: 800,000 L/mol. The value of KΔC; (i) accounts for the ability of O2 to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.

Three unknown quantities are all that is required to describe the O2-equilibrium binding curve for fractional saturation of human hemoglobin in red blood cells, under standard conditions: Kα, the O2-binding constant of equivalent α-chains; KC, the equilibrium constant for the T →R conformation change; Kβ, the O2-binding constant of equivalent β-chains. The model for formulation of the equation of state is a 3-stage ordered sequence of reactions. The values of were established by determination of rate constants for the oxygenation reaction and the dithionite-mediated de oxygenation reaction. The rate law for the forward reaction in the presence of excess O2 yields The same rate law yields for the dithionite-mediated de-oxygenation reaction. The rate constants for binding O2 are pseudo-first-order. The rate constants for release of O2 are first-order. Reactions involving O2, are 2-step ordered sequences of equivalent subunits. Progress curves for a 2-step ordered sequence of equivalent chains collapse to a first order reaction. Progress curves for both oxygenation and dithionite-mediated de-oxygenation reactions return is 0.0580 for the oxygenation reaction and 0.0358 for the dithionite-mediated de-oxygenation reaction. The corresponding values from the O2-equilibrium binding curve are: and = 0.02602. Values of determined from rate constants of progress curves for oxygenation and dithionite-mediated de-oxygenation reactions are close to values of determined by analysis of the O2-equilibrium binding curves for whole blood, by the Perutz/Adair equation.

Dual emission is a reality in many, but not all, ruthenium complex ions, even at room temperature and in fluid solution. It requires significantly different ligands and is more obvious in rigid media such as glasses at low temperature and crystalline powders. However, there are not just two unique lifetimes. Rather, there is a continuum of similar but slightly different lifetimes drawn from a bimodal distribution. In some of the cases that do not show dual emission, there still seems to be a continuum within a unimodal distribution. After reviewing a wide range of data, we describe possible interpretations. Most novel, but in our view most attractive, is a major role for ion pairing. If one admits that ions near charge transfer orbitals should have a stabilizing effect, and if one doubts that there would be any unique configuration for ion pairs, then it would seem that a continuous variation of excited state behavior is mandatory. This mechanism accounts for the fact that highly asymmetric substitutions at the 4-position of the phen ligand that most affects charge transfer always show dual emission and offer the most exaggerated differences between the two emissions. Other possibilities remain, given the data available at this time.

A family of dendritic tris-bipyridyl ruthenium coordination complexes incorporating two or four carboxylate groups for binding to a TiO(2) surface site and another dendritic linker between the metal complex and highly absorptive dyes were formulated as thin films on TiO(2) coated glass. The family included phenothiazine-substituted dendrons of increasing structural complexity and higher optical density. The dye-loaded films were characterized by steady-state emission and absorption measurements and by kinetic studies of luminescence and transient absorption. Upon photoexcitation of the bound dyes, rapid electron injection into the metal oxide film was the dominant observed process, producing oxidized dye that persisted for hundreds of milliseconds. Complex decay profiles for emission, transient absorption, and optical bleaching of the dendritic dyes point to highly heterogeneous behavior for the films, with observed persistence lifetimes related directly to structurally enhance electronic coupling between the metal oxide support and the dendritic dyes.

© 2015 Elsevier B.V. Dual emission is a reality in many, but not all, ruthenium complex ions, even at room temperature and in fluid solution. It requires significantly different ligands and is more obvious in rigid media such as glasses at low temperature and crystalline powders. However, there are not just two unique lifetimes. Rather, there is a continuum of similar but slightly different lifetimes drawn from a bimodal distribution. In some of the cases that do not show dual emission, there still seems to be a continuum within a unimodal distribution. After reviewing a wide range of data, we describe possible interpretations. Most novel, but in our view most attractive, is a major role for ion pairing. If one admits that ions near charge transfer orbitals should have a stabilizing effect, and if one doubts that there would be any unique configuration for ion pairs, then it would seem that a continuous variation of excited state behavior is mandatory. This mechanism accounts for the fact that highly asymmetric substitutions at the 4-position of the phen ligand that most affects charge transfer always show dual emission and offer the most exaggerated differences between the two emissions. Other possibilities remain, given the data available at this time.

Colloidal suspensions of 3-aminopropylmethyl(tetraphenyl)silole nanoparticles can be used as selective chemosensors for carcinogenic chromium(VI) analyte. Methylhydrosilole is functionalized by hydrosilation of allylamine, and the colloid is prepared by the rapid addition of water to a THF solution of the silole. The method of detection is through electron-transfer quenching of the fluorescence of the silole colloid (lambda(em) = 485 nm at 360 nm excitation) by the analytes, with hundred parts per billion detection limits. Stern-Volmer plots are linear up to 10 ppm in the case of chromium, but exhibit saturation behavior near 5-10 ppm for arsenic. Dynamic light scattering experiments and AFM measurements show the particle sizes to be around 100 nm in diameter and dependent on solvent composition, with a particle size dispersity of +/-25%. The fluorescence lifetimes of the silole in solution and colloid are approximately 31 ps and approximately 4.3 ns, respectively, while the silole has a lifetime of 6 ns in the bulk solid. A minimum volume fraction of 80% water is necessary to precipitate the colloid from THF, and the luminescence continues to rise with higher water fractions. Colloids in a pH 7 phosphate-buffered suspension show both higher sensitivity and greater selectivity (100-fold) for CrO4(2-) detection than for other oxoanion interferents, NO3-, NO2-, SO4(2-), and ClO4-.

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

Despite early claims that nitric oxide does not react with cobalamin under any circumstances, it is now accepted that NO has a high affinity for cobalamin in the 2+ oxidation state [Cbl(II)]. However, it is still the consensus that NO does not react with Cbl(III). We confirmed that NO coordinates to Cbl(II) at all pH values and that Cbl(III) does not react with NO at neutral pH. At low pH, however, Cbl(III) does react with NO by way of a two-step process that also reduces Cbl(III) to Cbl(II). To account for the pH dependence, and because of its intrinsic interest, we also studied reactions of NO with cobinamide [Cbi] in the 2+ and 3+ oxidation states. Both Cbi(II) and Cbi(III) react readily with NO at all pH values. Again, Cbi(III) is reduced during the process of coordinating NO. Compared to cobalamin, cobinamide lacks the tethered 5,6-dimethylbenzamidazolyl moiety bound to the cobalt ion. It may, therefore, be considered a "base-off" form of Cbl. To explain the reaction of Cbl(III) at low pH, we infer that the base-off form of Cbl(III) exists in trace amounts that are rapidly reduced to Cbl(II), which then binds NO efficiently. Base dissociation, we postulate, is the rate-limiting step. Interestingly, Cbi(II) has 100 times greater affinity for NO than does Cbl(II), proving that there is a strong trans effect due to the tethered base in nitrosyl derivatives of both Cbl(II) and Cbl(III). The affinity of Cbi(II) for NO is so high that it is a very efficient NO trap and, consequently, may have important biomedical uses.

The synthesis, spectroscopic characterization, and fluorescence quenching efficiency of polymers and copolymers containing tetraphenylsilole or tetraphenylgermole with Si-Si, Ge-Ge, and Si-Ge backbones are reported. Poly(tetraphenyl)germole, 2, was synthesized from the reduction of dichloro(tetraphenyl)germole with 2 equivs of Li. Silole-germole alternating copolymer 3 was synthesized by coupling dilithium salts of tetraphenylsilole dianion with dichloro(tetraphenyl)germole. Other tetraphenylmetallole-silane copolymers, 4-12, were synthesized through the Wurtz-type coupling of the dilithium salts of the tetraphenylmetallole dianion and corresponding dichloro(dialkyl)silanes. The molecular weights (M(w)) of these metallole-silane copolymers are in the range of 4000 approximately 6000. Detection of nitroaromatic molecules, such as nitrobenzene (NB), 2,4-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT), and picric acid (PA), has been explored. A linear Stern-Volmer relationship was observed for the first three analytes, but not for picric acid. Fluorescence spectra of polymetalloles or metallole-silane copolymers obtained in either toluene solutions or thin polymer films displayed no shift in the maximum of the emission wavelength. This suggests that the polymetalloles or metallole-silanes exhibit neither pi-stacking of polymer chains nor excimer formation. Fluorescence lifetimes of polymetalloles and metallole-silanes were measured both in the presence and absence of TNT, and tau(o)/tau is invariant. This requires that photoluminescence quenching occurs by a static mechanism.

Soluble guanylate cyclase (GC) from bovine lung is activated 4-fold by carbon monoxide (CO) and 400-fold by nitric oxide (NO). Spectroscopic and kinetic data for ligation of CO and NO with GC are summarized and compared with similar data for myoglobin (Mb), hemoglobin (Hb), and heme model compounds. Kinetic, thermodynamic, and structural data form a basis on which to construct a model for the manner in which the two ligands affect protein structure near the heme for heme proteins in general and for GC in particular. The most significant datum is that although association rates of ligands with GC are similar to those with Mb and Hb, their dissociation rates are dramatically faster. This suggests a delicate balance between five- and six-coordinate heme iron in both NO and CO complexes. Based on these and other data, a model for GC activation is proposed: The first step is formation of a six-coordinate species concomitant with tertiary and quaternary structural changes in protein structure and about a 4-fold increase in enzyme activity. In the second step, applicable to NO, the bond from iron to the proximal histidine ruptures, leading to additional relaxation in the quaternary and tertiary structure and a further 100-fold increase in activity. This is the main event in activation, available to NO and possibly other activators or combinations of activators. It is proposed, finally, that the proximal base freed in step 2, or some other protein base suitably positioned as a result of structural changes following ligation, may provide a center for nucleophilic substitution catalyzing the reaction GTP --> cGMP. An example is provided for a similar reaction in a derivatized protoheme model compound. The reaction mechanism attempts to rationalize the relative enzymatic activities of GC, heme-deficient GC, GC-CO, and GC-NO on a common basis and makes predictions for new activators that may be discovered in the future.

To study the dynamics and mechanism of intramolecular photoinduced electron transfer (PET) reactions, a series of (ZnII−FeIII) meso-tetraarylmetalloporphyrin dimers were synthesized and the kinetics of their PET reactivity was measured. Molecular building blocks were prepared by selective nucleophilic aromatic substitution of a para fluorine on tetraarylporphyrins containing a single pentafluorophenyl group. This synthetic approach allows a wide variety of systematic modifications such as type and length of spacer, metal center, and redox-potential difference between donor and acceptor. The edge-to-edge distance between the two porphyrins varies from 14.4 to 27.3 A. Into a symmetric dimer, with two identical porphyrins covalently linked by a rigid partly saturated bridge, one zinc(II) and one iron(III) can be inserted. From measurements of fluorescence lifetimes the rate constants for PET from the electronically excited state of the zinc porphyrin to the bis(imidazole)iron porphyrin cation were evaluated....

Fluorescence lifetimes of five representative xanthene dye species-the rhodamine B zwitterion (RB'), the rhodamine B cation (RB+), the rhodamine 66 cation (R6G+), the rhodamine 101 zwitterion (R101') and the fluorescein dianion (F2-)-were measured in HzO, D20 and in a series of alcohol solvents ranging from methanol to octanol. The lietimes of both RB* and RB+ increased markedly as the solvent was varied from water to actanol. In contrast, the lifetimes of R6G+ and R101' decreased slightly over the alcohol series and that of F2- increased only slightly in the same series. For all the dyes studied the fluorescence lifetimes observed in DzO were slightly longer than those in H20. Possible causes for the variations observed are discussed.

Kinetics of NO dissociation were characterized for three five-coordinate systems, heme-NO, HSA-heme-NO (human serum albumin), GC-NO (soluble guanylate cyclase), and for the six-coordinate system, Im-heme-NO. Nitrosyl myoglobin was redetermined for comparison. Previously known, six-coordinate R and T state nitrosyl hemoglobins are also included in the comparison. The data indicate that NO dissociates more than 1000 times faster from five-coordinate model heme than it does from the six-coordinate analog. Such a negative trans-effect between NO and a proximal base is in sharp contrast to carboxy heme derivatives, in which ligand dissociation rates are greatly slowed in when a trans base is present. As a result of opposite trans-effects, six-coordinate carboxy and nitrosyl derivatives have comparable dissociation rates, even though the five-coordinate species are very different. In proteins, five- and six-coordinate forms do not show a large difference in dissociation rates. Part of the reason may be due to different probabilities for geminate recombination in the different proteins, but this cannot explain all the facts. There must also be influences of the protein structure on bond-breaking rate constants themselves. With the exception of hemoglobin in the T state, nitrosyl guanylate cyclase shows the highest NO dissociation rate constant, k(obs) = 6 x 10(-4) s(-1). This would yield a half-life of about 2 min at 37 degrees C for dissociation of NO from GC-NO, a number that has implications for the mechanism of regulation of the activity of this key heme enzyme.

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce .NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 10(6) M-1 s-1) and another slow (2-4 x 10(4) M-1 s-1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, approximately 50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s-1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

The emission of the merocyanines derived from the photochromic 1, 3, 3-trimethylspiro[indoline-2,3′[3H]naphth[2,1-b][1, 4]oxazine] 1, the 4, 5-dimethyl-substituted compound 2 and the 6′-piperidino-substituted derivative 3, in solution with acetonitrile or ethanol, was studied by means of a time-correlated single photon counting system with ca 10 ps time resolution. The signal decays were described by a sum of two or three exponential components with τ1=10–60 ps, τ2= 0.5–1ns, τ3=1–2 ns, corresponding to the contribution of different fluorescing merocyanine isomers. The most intense emission, the shortest lived one, is characterized by extremely low quantum yields (

In either sperm whale or horse heart myoglobin, binding of NO and lowering of solution pH work together to weaken, and ultimately break, the bond between iron and the proximal histidine. This is reminiscent of the reaction observed at neutral pH in the case of guanylate cyclase, the heme enzyme that catalyzes the conversion of GTP to cGMP. Bond breaking is characterized by a spectral change from a nine-line to a three-line ESR signal and accompanied by a shift from 420 to 387 nm in the UV-vis spectrum of the Soret band maximum. Analysis of the pH-dependent spectral changes shows that they are reversible, at least within a few hours, that the transition is cooperative, involving six protons during pH lowering but only two as it is raised, and that the pK is about 4.7. Different proteins exhibit different pK values, which are generally lower than that for "chelated" protoheme. The pK differences reflect the extra bond stability afforded by the protein structure. Investigations of thermal and photochemical NO displacement by CO suggest that the local pocket around the ligand, although significantly altered (according to circular dichroism investigations), nonetheless still imposes a barrier against the outward diffusion of ligand into the solvent. Nanosecond and picosecond flash photolysis shows that in proteins at low pH there is an extremely efficient geminate recombination of the ligand with the four-coordinated species through a single-exponential process. This occurs to a significantly larger extent than for the case of NO-"chelated" protoheme (where no distal barrier for ligand is present). At neutral pH, when the proximal histidine bond is intact, the geminate recombination for NO takes longer and displays multiexponential kinetics. Altogether, these results suggest that, even though distal effects probably also play a role, proximal effects make an important contribution in modulating ligand-iron bond formation.

The quantum yields for photolysis of imidazole-iron(II) porphyrin-NO complexes have been determined in the following solvents: toluene, aqueous 1-methylimidazole, Nujol, and glycerol. The corresponding yields are 0.2, 0.077 (30% 1-methylimidazole in water), 0.064, and 0.007. Steric effects on NO photolysis were also investigated. The increasingly hindered porphyrins (mesoheme, anthracene-7,7-cyclophane heme, adamantane-6,6-cyclophane heme, and pyridine-5,5-cyclophane heme) had, in toluene, quantum yields of 0.21, 0.21, 0.29, and 0.49, respectively. Several conclusions are drawn from these results. (1) Polar solvents decrease NO quantum yields

Kinetics and equilibria of binding of 1-methylimidazole, 1,2-dimethylimidazole, isonitriles, and carbon monoxide to iron(II) tetraphenylporphyrin and iron(II) tetramesitylporphyrin bearing various ligands in the sixth position have been determined.

Two different ΔS=2 intersystem crossing processes, the 1 MLCT→ 5 T 2 and 5 T 2 → 1 A 1 conversions which follow the photoexcitation of low-spin Fe II complexes, are examined on the nanosecond, picosecond, and subpicosecond time scales

Boron difluoride adducts of diamidodipyrromethenes have been synthesized and characterized. The compounds represent a new group of the BODIPY family of fluorescent dyes. X-ray crystallography and solution (19)F NMR experiments show that a persistent hydrogen bond is formed between the boron-bound fluoride groups and the peripheral amide substituents. The modular synthesis of these compounds and their robust photophysical properties suggest that they may be useful compounds for materials and biological photochemical applications.

The kinetics of transient absorption changes for a number of protoheme-ligand systems after subpicosecond photolysis have been investigated. When the photolyzed ligand is tert-butyl, pentachlorophenyl, pentafluorophenyl, 5α-cholestan-3α-yl, or 5α-cholestan-3β-yl isocyanide or 1-methylimidazole, a concentration-independent relaxation is observed. Its decay is accuratel exponential. Therefore the geminate pair created by photolysis disappears in a clear first-order process and does not follow the power law kinetics reported for similar systems with other ligands in high-viscosity solvents or in glasses at low temperatures

The picosecond photolysis of protoheme dimethyl ester bis(tert-butyl isocyanide) in pure tert-butyl isocyani in toluene results in loss of one isocyanide to produce an electronically excited five-coordinated species. The excicited state decays to ground-state five-coordinated heme within 40 ps. In contrast to chelated protoheme-tert-butyl isocyanide or imidazole-protoheme dimethyl ester-tert-butyl isocyanide, no cage return is seen.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1–2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.

The effect of pressure on the recombination kinetics of small ligands binding to sperm whale myoglobin, protoheme dimethyl ester, and monochelated protoheme was studied with use of laser flash photolysis. The volumes of activation observed indicate that in both the protein and the models bond formation is the rate-determining step only for carbon monoxide, while for oxygen, isocyanides, and 1-methylimidazole almost no bond formation occurs in the transition state of the observed reaction. The effect of pressure on the escape of carbon monoxide, oxygen, and methyl isocyanide from the heme pocket of sperm whale myoglobin was also investigated

Steric effects on the binding of isonitrile ligands to iron(II) porphyrins were investigated by picosecond flash photolysis. Two different types of steric effects were distinguished and characterized: (1) steric restrictions to porphyrin planarity and (2) blocking of the pathway for ligand approach. Heme planarity was restricted by coordinating 1,2-dimethylimidazole trans to the ligand binding site being investigated. Blocking of the binding site was explored by using adamantane heme 6,6-cyclophane, in which the adamantane moiety forms a cap over the binding site. Results of picosecond kinetic measurements demonstrate that the first effect, heme nonplanarity or trans strain, influences the bond-making step, whereas the second effect, ligand blocking, involves a conformational preequilibrium prior to bond making. Relevance of these findings for contact pair recombination, in general, and for heme protein ligation, in particular, are discussed.

The kinetics of geminate recombination were studied for the methylisocyanide derivative of carp hemoglobin. Carp hemoglobin is of interest because it has been established that the fully liganded form switches between a high affinity R state at pH 9 and a low affinity T state at pH 6 in the presence of IHP. Geminate recombination was observed on both the picosecond and the nanosecond time scales under all conditions; however, only a small variation is observed in the rates and the yields of geminate recombination as the protein switches from the R to the T state. Taken together with overall "on" and "off" rates, the data indicate that the change from the R to the T configuration affects bond breaking most, but also influences subsequent escape from the protein as well as both entry into the protein and bond formation. There is some reason to postulate tertiary conformational change in the T state on the microsecond time scale following ligand escape from the protein.

A family of heteroleptic RuII coordination complexes containing substituted 1,10-phenanthroline (phen) ligands with extended conjugation was found to exhibit two simultaneously emissive excited states at room temperature in fluid solution. These systems demonstrate a breakdown of the standard non-radiative decay pathways that normally lead to a single, dominant, lowest energy emissive excited state in RuII complexes and most other chromophores. The structural requirements for dual emission were explored through the synthesis and characterization of isomeric systems. Two features were found to be primarily responsible for resolvable dual emission. Extended conjugation at the 4-position of the 1,10-phenanthroline ligand was identified as an essential feature, and asymmetry in the phenanthroline ligand substitutions appears to greatly facilitate the production of these two non-equilibrated emissive states. Additional complexes were studied which displayed “tunable” emissive characteristics for the two excited states as a function of covalent and non-covalent modification.

Colloidal oligo(tetraphenyl)silole nanoparticles in THF/H2O suspensions show increased luminescence and offer a method to detect TNT in an aqueous environment.

Tris(bipyridyl)ruthenium(II) complexes modified such that one of the bipyridines is appended with a crown ether display luminescence that is responsive to complexation with metal ions. The parent species, Ru(bpy)3(2+), is moderately luminescent, with an emission lifetime of about 1 micros in fluid solution at room temperature. The modified complexes are much less emissive, with lifetimes near 1 ns. Conformational flexibility and distortion in the crown-ether complexes enhance nonradiative decay. Noncovalent binding of metal ions, however, restores luminescence intensity by reducing nonradiative decay and increasing the lifetime 10- to 100-fold. Reported here are the syntheses and steady-state and time-resolved luminescence measurements in addition to other supporting spectroscopic characterization. Seven metals were investigated; significant luminescence enhancements occur in the presence of Mg2+, Ca2+, and Pb2+. Effective concentrations of metal ions range from tens of microM to hundreds of mM. The steady-state enhancements are readily measured, but they are less than would be expected from the lifetime changes, partly because only a portion (not more than 50%) of the fast (1 ns) decay in Ru(bpy)2(bpy-crown) is capable of converting to the conformation possessing the longer lifetime. A photophysical model is proposed to explain these and other observations.

Dual emission is observed from a family of simple acetylene-linked dinuclear RuII complexes, where two MLCT excited states coexist at room temperature and in fluid solution. This unique behavior is attributed to a specific substitution pattern on the bridging ligand and provides the opportunity to investigate the structural and electronic features that result in decoupling of standard nonradiative decay pathways.

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

Oxygenase and reductase domains in nitric oxide synthase are linked by a peptide region that binds calmodulin. Here we study the effects of modifying the length of the interdomain linker in a deletion mutant lacking 15 amino acids (residues 503-517) in bovine eNOS. The kinetics of CO ligation with the mutant were determined in the presence and absence of tetrahydrobiopterin and arginine and compared with the CO binding kinetics of wild-type eNOS and the eNOS oxygenase domain. In the mutant, electron flow is interrupted. The association kinetics of CO with both mutant and wild-type eNOS can be approximated with two kinetic phases, but the relative proportions change in the mutant. Both the abrogation of electron flow in the mutant and the differences in CO binding may be explained by an alteration in the docking of the FMN domain to the heme domain. We propose that the calmodulin binding residues form a helix that is critical for the proper alignment of the adjacent reductase and oxygenase domains within the active eNOS dimer in achieving proper electron transfer between them.

Absolute fluorescence quantum yields are reported for the rhodamine 6G cation and the fluorescein dianion dyes in nine solvents. This information is combined with previously reported fluorescence lifetimes to deduce radiative and nonradiative decay rates. Along the alcohol series from methanol to octanol, rhodamine 6G displays an increasing radiative rate, in parallel with the square of the refractive index increase, and a slightly decreasing nonradiative rate. Fluorescein is different: the apparent radiative rate actually decreases, suggesting that the emissive species is perturbed in some fashion. For both dyes, fluorescence yields are enhanced in D2O, rising to 0.98, in parallel with a corresponding increase in lifetimes. Protonated solvents invariably give shorter lifetimes and lower quantum yields, contrary to some previous speculation. From this work and an analysis of existing literature values, more precise values have been obtained for two previously proposed absolute quantum yield standards. The yield of fluorescein in 0.1 N NaOH(aq) is 0.925+/-0.015, and for rhodamine 6G in ethanol, it is 0.950+/-0.015. In both cases, the solutions are assumed to be in the limit of low concentration, excited close to their long-wave absorption band and at room temperature but may be either air-saturated or free of oxygen.

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.

The synthesis of caged NADP analogues 18, 19, and 20 has been accomplished by utilizing the transglycosidase activity of solubilized NAD glycohydrolase (porcine brain) to incorporate caged nicotinamides 2, 3, and 4 into NADP. The synthesis of several nicotinamides modified at the carboxamide with o-nitrobenzyl photolabile groups is demonstrated as well as their potential for enzymatic transglycosidation. These results further demonstrate the feasibility of direct enzymatic transglycosidation of sterically hindered substrates into NAD(P), although high nicotinamide analogue water solubility was found to be a necessary trait for yield enhancement with certain analogues. Caged analogues were surveyed under aqueous conditions for net NADP photorelease, while the UV and fluorescent properties of both analogues and their photobyproducts were assessed for compatibility with systems that rely on optical monitoring of enzyme activity. A highly water-soluble alpha-methyl-o-nitrobenzyl group 8 was developed for the synthesis of 20 in order to enhance net NADP photorelease. Compound 20 demonstrated a high 75% net NADP photoreleased without substantial UV optical blackening or fluorescent byproducts. Analogues 18 and 19 were shown by ESI/MALDI-MS to photogenerate primarily adducts of NADP with deleterious UV and fluorescent properties. Our work stresses the superior release properties conferred by alpha-methyl substitution on aqueous carboxamide photorelease from o-nitrobenzyl compounds.

Previous work has proved that the enzyme-soluble guanylate cyclase, GC, is activated several 100-fold by the combination of carbon monoxide plus a benzylindazole derivative called YC-1. That is about the same as activation by nitric oxide, which has a well-established role both in vivo and in vitro. This report addresses several spectroscopic, equilibrium, and kinetic effects wrought by YC-1 on carboxyl guanylate cyclase, including the following: a shift in the Soret absorption band by 4 nm to shorter wavelength; an increase in CO affinity by an order of magnitude; a dramatic change in the kinetics of CO association. After photolytic dissociation of CO, the majority, but not all, of bimolecular ligand recombination occurs with a time constant about 1000-fold faster than in the absence of YC-1, while a smaller fraction recombines almost, but not quite, the same as usual. This is reminiscent of the kinetics of NO association with GC, which also shows two prominent phases. The results just listed pertain in the presence of GTP/cGMP, which would be present during enzyme catalysis. Qualitatively similar, but smaller, effects occur in the absence of GTP/cGMP. Measurements are reported to characterize other changes in buffer conditions. The results are consistent with a mechanistic model that attributes a crucial role to the proximal bond that connects the heme iron to a histidine side chain in GC but also requires protein control of the distal environment.

Nitric-oxide synthase (NOS) catalyzes conversion of L-arginine to nitric oxide, which subsequently stimulates a host of physiological processes. Prior work suggests that NOS is inhibited by NO, providing opportunities for autoregulation. This contribution reports that NO reacts rapidly (ka congruent with 2 x 10(7) M-1 s-1) with neuronal NOS in both its ferric and ferrous oxidation states. Association kinetics are almost unaffected by L-arginine or the cofactor tetrahydrobiopterin. There is no evidence for the distinct two phases previously reported for association kinetics of CO. Small amounts of geminate recombination of NO trapped in a protein pocket can be observed over nanoseconds, and a much larger amount is inferred to take place at picosecond time scales. Dissociation rates are also very fast from the ferric form, in the neighborhood of 50 s-1, when measured by extrapolating association rates to the zero NO concentration limit. Scavenging experiments give dissociation rate constants more than an order of magnitude slower: still quite fast. For the ferrous species, extrapolation is not distinguishable from zero, while scavenging experiments give a dissociation rate constant near 10(-4) s-1. Implications of these results for interactions near the heme binding site are discussed.

Recently it has been reported that in the presence of YC-1, a benzyl indazole derivative, carbon monoxide activates soluble guanylate cyclase, GC, to about the same extent as its best known activator, nitric oxide. Kinetic studies utilizing flash photolysis of GC complexed with CO in the presence and absence of YC-1 show, in contrast to another recent report of a mixing experiment, that YC-1 has a profound effect on bimolecular association kinetics and a smaller, but significant, effect on ligand affinity. Most prominent is the appearance of a major, new phase in the bimolecular recombination kinetics in the presence of 200 microM YC-1: This major fraction rebinds CO approximately 1000-fold more rapidly than in the absence of YC-1. Another portion, considerably less than half, exhibits kinetics that are almost exactly the same as in the absence of YC-1. It is now clear that both YC-1 and CO have a strong synergistic effect on enzyme activity and also a dramatic effect on ligand binding behavior. It is, therefore, a reasonable inference that ligand binding at the heme iron atom is intimately connected with enzyme activation, a hypothesis that would have been difficult to maintain if the earlier report, that YC-1 has no effect on CO binding, were correct. Possible reasons for the discrepancy between the two measurements are suggested.