Dichroism Bands Research Articles

Pick and Choose the Coordinated Metal Ion to Tune the Porphyrin Supramolecular Chirogenesis

AbstractThe solvent‐driven aggregation of metalloporphyrin derivatives, bearing an L‐ or D‐prolinate appendage, results in the stereospecific formation of chiral species that are strictly influenced by the aminoacid chirality and, above all, by the metal axial coordination capability. The Pd porphyrin derivatives self‐assemble in hydroalcoholic mixtures in fractal‐like suprastructures, displaying intense, specular dichroic bands and peculiar UV‐Vis spectral features, accounting for the formation of J‐type aggregates of complex morphology. Spectroscopic and kinetic studies gave important information on the aggregation process, allowing for a comparison with previously reported systems achieved from the free base and Zn derivatives in the same aggregative conditions. Analyzing the self‐aggregation process for the three porphyrin derivatives highlights the remarkable influence of porphyrin inner core features on the chirogenesis of the final systems, revealing an easy tunability of the asymmetry of the assemblies upon metal choosing.

Simons Observatory Focal-Plane Module: In-lab Testing and Characterization Program

The Simons Observatory is a ground-based cosmic microwave background instrument to be sited in the Atacama Desert in Chile. SO will deploy 60,000 transition-edge sensors (TES) bolometers in 49 separate focal-plane modules across a suite of four telescopes covering three dichroic bands termed low frequency (LF), mid-frequency and ultra-high frequency. Each MF and UHF focal-plane module packages 1720 feedhorn-coupled detectors with cryogenic components for highly multiplexed readout using microwave SQUID multiplexing. In this paper, we describe the testing program we have developed for high-throughput validation of modules after they are assembled. The validation requires measurements of the yield, saturation powers, time constants, noise properties and optical efficiencies. Additional measurements will be performed for further characterization as needed. We describe the methods developed and demonstrate preliminary results from the initial testing of a prototype module.

Absolute Configuration Sensing of Chiral Aryl- and Aryloxy-Propionic Acids by Biphenyl Chiroptical Probes

The absolute configuration of chiral 2-aryl and 2-aryloxy propionic acids, which are among the most common chiral environmental pollutants, has been readily and reliably established by either electronic circular dichroism spectroscopy or optical rotation measurements employing suitably designed 4,4′-disubstituted biphenyl probes. In fact, the 4,4′-biphenyl substitution gives rise to a red shift of the diagnostic electronic circular dichroism signal of the biphenyl A band employed for the configuration assignment, removing its overlap with other interfering dichroic bands and allowing its clear sign identification. The largest A band red shift, and thus the most reliable results, are obtained by employing as a probe the 4,4′-dinitro substituted biphenylazepine 3c. The method was applied to the absolute configuration assignment of 2-arylpropionic acids ibuprofen (1a), naproxen (1b), ketoprofen (1c) and flurbiprofen (1d), as well as to the 2-aryloxypropionic acids 2-phenoxypropionic acid (2a) and 2-naphthoxypropionic acid (2b). This approach, allowing us to reveal the sample’s absolute configuration by simple optical rotation measurements, is potentially applicable to online analyses of both the enantiomeric composition and absolute configuration of these chiral pollutants.

Theoretical VCD response in the C-H stretching region of methyl α and β L-Fucopyranoside: a different behavior from monosaccharides

Vibrational circular dichroism (VCD) band near 2840 cm−1 in the C-H stretching region is likely to play an important role as an alternative approach to extract stereochemical information namely, glycosidic O-linkages α or β for monosaccharides. Indeed, the experimental results attribute a positive sign for the S absolute configuration (α-anomers for D-sugars and β-anomers for L-sugars) and a negative sign for the R absolute configuration (β-anomers for D-sugar and α-anomers for L-sugar) at C-1 anomeric carbon site. This band was assigned as a symmetric stretching motion of glycosidic methyl group. The idea was to reproduce and explain these experimental results by using theoretical powerful methods and go farther, by studying how to extract stereochemical information from polysaccharides. The obtained theoretical VCD spectra for six monosaccharides were in good agreement with the experiment, except for methyl L-Fucopyranoside. Truly, for this particular sugar, two VCD bands with different signs were obtained for the S-configuration (β form) and assigned both to the symmetric stretching of the methyl glycosidic group. It becomes more interesting to explore this VCD response, to allow us, possibly to examine another way to differentiate between the alpha and the beta forms for this molecule.

Learning how planarization can affect dichroic patterns in polyfluorenes.

The circular dichroism spectra of a single chain of polyfluorene was predicted for a p-twisted helix conformation and local planarized polymer sections in the presence and in the absence of thermal vibrations. Under thermal vibrations at 300 K, the planarized section of polyfluorene affords a red-shifted positive dichroic band between 446 and 456 nm, preserving a degree of chirality. The S1 ← S0 excitation energy decreases from 3.29 eV, for the p-twisted helix to 2.77 or 2.71 eV, for planarized sections with one or two coplanar twists, respectively. Thermal vibrations and intramolecular rotations eventually affect the circular dichroism spectrum patterns, where planarized bent conformers induce a positive band towards 450 nm.

Helical Assembly of Flavin Mononucleotides on Carbon Nanotubes as Multimodal Near-IR Hg(II)-Selective Probes.

The development of novel methods to detect mercury is of paramount importance owing to the impact of this metal on human health and the environment. We observed that flavin mononucleotide (FMN) and its helical assembly with a single-walled carbon nanotube (SWNT) selectively bind Hg2+ arising from HgCl2 and MeHgCl. Absorption spectroscopic studies show that FMN preferentially forms a 2:1 rather than a 1:1 complex with Hg2+ at high FMN concentrations. On the basis of the analogy to the thymine-Hg-thymine complex, it is proposed that the 2:1 complex between FMN and Hg2+ comprises a Hg-bridged pair of FMN groups, regardless of the presence of SWNT. Upon addition of as little as a few hundred nanomoles of Hg2+, both FMN and FMN-SWNT exhibit absorption and photoluminescence (PL) changes. Moreover, FMN-SWNT displays simultaneous multiple sigmoidal changes in PL of SWNT tubes having different chiral vectors. Assessment of binding affinities using the Hill equation suggests that 2:1 and 1:1 complexes form between Hg2+ and FMN groups on the FMN-SWNT. Theoretical calculations indicate that optical changes of the FMN-SWNT originate from Hg-mediated conformational changes occurring on the helical array of FMN on the SWNT. High-resolution transmission electron microscopy revealed that the presence of Hg2+ in complexes with the FMN-SWNT enables visualization of helical periodic undulation of FMN groups along SWNT without the need for staining. Circular dichroism (CD) study revealed that FMN-SWNT whose CD signal mainly originates from FMN decreases dichroic bands upon the addition of Hg2+ owing to the formation of a centrosymmetric FMN-Hg-FMN triad on SWNT. The binding mode specificity and multimodal changes observed in response to Hg2+ ions suggest that systems based on FMN-SWNT can serve as in vivo NIR beacons for the detection of various mercury derivatives.

Influence of Chitosan Ascorbate Chirality on the Gelation Kinetics and Properties of Silicon-Chitosan-Containing Glycerohydrogels.

The influence of the chirality of chitosan ascorbate on the gelation kinetics and the properties of hybrid silicon-chitosan-containing glycerohydrogels were studied with a deep estimation of the stereospecificity of chitosan polysalts with l- and d-ascorbic acid diastereomers and their biological effects. It has been established that l- and d-diastereomerically enriched chitosan ascorbates are characterized by a positive Cotton effect and differ in the wavelength of the maximum of the dichroic band (250 and 240 nm), as well as in the values of its specific ellipticity (21.8 × 105 and 39.2 × 105 deg·mL·dm−1·g−1), the sign of specific optical rotation (+ and −), the type of dispersion curves (anomalous and smooth), as well as the condensed phase morphology (anisodiametric particles with optical anisotropy and confocal domains of spherical shape, respectively). In the biomimetic sol-gel synthesis of silicon-chitosan-containing glycerohydrogels using silicon tetraglycerolate as a precursor, it was found that chitosan d-ascorbate retarded gelation. Thin congruent plates obtained from the corresponding glycerohydrogels based on chitosan d-ascorbate have higher mechanical strength and elasticity under uniaxial stretching and lower values of Young’s modulus. It has been shown that the systems based on chitosan d-ascorbate show the greatest antibacterial activity against Staphylococcus aureus 209P and Escherichia coli 113-13 and significantly promote the viability of normal human dermal fibroblasts. The results of our assessment of the biological properties of chitosan polysalts are unexpected, since ascorbic acid exhibits biological activity as its l-isomer only.

Orientation of non-spherical protonated water clusters revealed by infrared absorption dichroism

Infrared continuum bands that extend over a broad frequency range are a key spectral signature of protonated water clusters. They are observed for many membrane proteins that contain internal water molecules, but their microscopic mechanism has remained unclear. Here we compute infrared spectra for protonated and unprotonated water chains, discs, and droplets from ab initio molecular dynamics simulations. The continuum bands of the protonated clusters exhibit significant anisotropy for chains and discs, with increased absorption along the direction of maximal cluster extension. We show that the continuum band arises from the nuclei motion near the excess charge, with a long-ranged amplification due to the electronic polarizability. Our experimental, polarization-resolved light–dark difference spectrum of the light-driven proton pump bacteriorhodopsin exhibits a pronounced dichroic continuum band. Our results suggest that the protonated water cluster responsible for the continuum band of bacteriorhodopsin is oriented perpendicularly to the membrane normal.

Programmable Extreme Chirality in the Visible by Helix-Shaped Metamaterial Platform.

The capability to fully control the chiro-optical properties of metamaterials in the visible range enables a number of applications from integrated photonics to life science. To achieve this goal, a simultaneous control over complex spatial and localized structuring as well as material composition at the nanoscale is required. Here, we demonstrate how circular dichroic bands and optical rotation can be effectively and independently tailored throughout the visible regime as a function of the fundamental meta-atoms properties and of their three dimensional architecture in a the helix-shaped metamaterials. The record chiro-optical effects obtained in the visible range are accompanied by an additional control over optical efficiency, even in the plasmonic context. These achievements pave the way toward fully integrated chiral photonic devices.

Existence of hydroxylated MWCNTs demotes the catalysis effect of amylases against starch degradation

Possible interaction between amylase and Multi Walled Carbon Nanotubes (MWCNTs) has been elucidated with spectroscopic methods. Hyperchromism of the UV⿿visible spectra of amylase-CNT conjugates suggested ground state complex formation between them. On contrary, the decreasing fluorescence emission spectra revealed the fate of quenching mechanism to be static. Stoke shift observed from the synchronous and 3D spectra suggested the possibilities of disturbances to the aromatic micro-environment of amylases by OH-MWCNTS. FTIR and FT-Raman spectra showed alteration in the amide I band, that corresponds to their effect on alpha-helical structures. Loss of alpha-helical structures and alteration in the dichroic band again revealed possible conformational change and effect towards the stability of polypeptide backbone structures. In addition, the shift observed in the SPR band and FTIR peaks of CNTs-amylase conjugates suggested possible alteration in their optical and structural properties. On the functional aspect, amylase activity on starch degradation and hydrolysis were found to be decreased in the presence of CNTs.

Adjustable spatial resolution of compressive spectral images sensed by multispectral filter array-based sensors

Spectral imaging systems capture spectral and spatial information from a scene to produce a spectral data cube. Technical progress has allowed developing multispectral filter array (MSFA)-based sensors in order to expand the reconstruction of more bands than RGB cameras. However, reconstructing the spectral image with traditional methods following a least squares or demosaicing approach is unfeasible. Some works in the literature implement multispectral demosaicing for reconstructing images with specific spatio-spectral resolution depending on the number of pixels in the detector and the filter mosaic. Recently, compressive sensing technique has been developed that allows reconstructing signals with fewer measurements than the traditional methods by using the sparse representation of a signal. The selection of neighborhoods pixels in the MSFA-based sensor to calculate the spectral response of a single pixel in the reconstructed spectral images could improve the reconstruction, based on exploiting the sparse representation of the spectral images. This paper proposes two models for spectral images reconstruction from the selection of MSFA-based sensor measurements neighborhoods using the principle of compressive sensing. The spatial resolution of the reconstructed spectral images is adjusted depending the size of the neighborhood. To verify the effectiveness of the reconstruction models simulated measurements for synthetic spectral images and real spectral images based on MSFA are used. Ensembles of random dichroic and random band pass filters are used. The two approaches with traditional scheme reconstructions of mosaic filters are compared. The proposed methods improve the quality (PSNR) of the image reconstruction up 7 dB for real spectral images.

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Biaxial and antiferroelectric structure of the orthogonal smectic phase of a bent-shaped molecule and helical structure in a chiral mixture system

We examined the biaxial and antiferroelectric properties in the Smectic-AP(A) (Sm-AP(A)) phase of bent-shaped DC-S-8. The biaxiality, which results from the existence of a secondary director, was well established from birefringence observations in the homeotropically aligned Sm-AP(A). By entering into Sm-AP(A) phase, the birefringence (Δn, difference between two refractive indices of short axes) continuously increased from 0 to 0.02 with decreasing temperature. The antiferroelectric switching and second harmonic generation (SHG) activity on the field-on state were also observed in the Sm-AP(A) phase, and the evaluated spontaneous polarization (P(S)) value strongly depended on temperature. The temperature dependence of Δn and P(S) resembles each other and follows Haller's approximation, showing that the biaxiality is due to polar packing in which the molecules are preferentially packed with their bent direction arranged in the same direction, and that the phase transition of Sm-AP(A) to Sm-A is second order. The biaxiality was further examined in chiral Sm-AP(A)(*). Doping with chiral components induced the helical twisting of the secondary director in the Sm-AP(A)(*) phase, which was confirmed by observing the reflection of the circular dichroism (CD) bands in the homeotropically aligned cell. The helical pitch of Sm-AP(A)(*) is tunable in the range of 300-700 nm wavelength with a variation in the chiral content of 5 to 10 weight (wt)%.

Ruthenium Red Colorimetric and Birefringent Staining of Amyloid-β Aggregates in Vitro and in Tg2576 Mice

Alzheimer's disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-β (Aβ) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aβ fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aβ aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aβ deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aβ plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aβ plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aβ fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aβ fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aβ aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Implementation of High Dynamic Raman Lidar System for 3D Map of Particulate Optical Properties and Their Time Evolution

Fast scanning lidar system can provide aerosol volume distribution and time evolution in atmosphere. Usually fast scanning lidar has a relative simple configuration. Multiwavelength depolarization and Raman measurements gave us more information about the particulate shape, type and dimension. The most Raman lidars use high power laser source in order to get enough Raman scattering signal. One of a challenge for Raman lidar system is to have enough dynamic range to satisfy the measurements both for pure molecule Rayleigh scattering and high dene aerosol, such as dust storm, volcano emitted aerosol and high polluted urban aerosol. The estimation of microphysical properties requires independent measurements of both backscatter and extinction coefficient at several wavelengths (multiwavelength Raman lidar). Additional information can be retrieved from simultaneous measurements of the depolarization signal and water vapor mixing ratio, since those measurements are particularly useful to correlate aerosol optical properties with their shape and hygroscopicity. A multi-wavelength depolarization and Raman lidar system has been designed to perform volume scanning of the atmosphere and to retrieve high quality 3D map of particulate optical properties and their time evolution. This system is equipped with a doubled and tripled Nd:YAG diode-pumped laser that is specifically designed for this device, with a repetition rate of 1KHz and average optical power of 0.6W at 355nm, 1.5W at 532nm and 1W at 1064nm. The relative high repetition rate laser source can increase the detectable signal dynamic range. The receiving system is based on a 25cm modified Cassegrain telescope. The spectral selection of the backscatter elastic and Raman signals is made through a system of dichroic beam splitters and narrow band (0.5 nm) interferential filters. Fast single photon counting photomultipliers are used to collect the selected radiation. Each detected signal is acquired by multichannel scalers with a raw spatial resolution varying from 30cm to 30m. Moreover, polarization purity of laser line allows to perform polarization measurements at both 355 and 532nm. This device is installed in the Beijing city area, which is strongly affected from anthropogenic pollution and sand dust from Gobi desert.

Phenoxathiinsulphone derivatives–cyclodextrin interactions: induced chirality and TDDFT calculations

The inclusion process of phenoxathiin-10,10-dioxide and 2-CH2Br-phenoxathiin-10,10-dioxide in α-, β-, γ- and 2-hydroxypropyl-γ-cyclodextrins was studied by circular dichroism spectroscopy. The dependence of the induced circular dichroism signal on the host concentration was analyzed in terms of a nonlinear model yielding the stoichiometry and the association constants of the complexes. Time dependent density functional theory (TDDFT) calculations were used to rationalize the experimental data considering two aspects. Firstly, to support on theoretical grounds the experimentally observed achirality of the studied compounds that present two structural elements to confer chirality: the butterfly motion of the roof-shaped heteroring and the rotation of the CH2Br group. In this last process, some favorable position of the bromine atom could influence the overall chirality. Secondly, the TDDFT calculations of the polarizations of the electronic transitions in correlation with the signs of the induced dichroic bands were used to establish the axial or equatorial way the guest is included in the host cavity.

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.

Intensities of circular dichroism and absorption bands and the states of d ions in absorbing media: II. Tetrahedrally coordinated positions of d ions

The factors affecting the band intensity in the circular dichroism (CD) and absorption spectra of tetrahedrally coordinated d ions in an absorbing medium (symmetry selection rules, structural position, and bond covalence) are analyzed. It is shown by the examples of the Cr4+ ion in Ca3Ga2Ge4O14 crystal and the Fe2+ and Fe3+ ions in SiO4 crystal that the symmetry forbiddenness of the transitions from orbitally degenerate states and the covalence of the d-ion-ligand bond lead to changes in the intensity of the corresponding CD bands in a wide range, beginning from zero. It is shown by the example of Ca3Ga2Ge4O14, LiAlGeO4, LiGaGeO4, and LiGaSiO4 crystals activated with Cr4+ ions that the preferred ion localization position corresponds to a higher effective symmetry.

Arg‐85 and Thr‐430 in murine 5‐aminolevulinate synthase coordinate acyl‐CoA‐binding and contribute to substrate specificity

5-Aminolevulinate synthase (ALAS) controls the rate-limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl-coenzyme A and glycine to produce 5-aminolevulinate, coenzyme-A (CoA), and carbon dioxide. ALAS is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X-ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl-CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl-CoA-binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl-CoA binding and substrate discrimination were examined using site-directed mutagenesis and a series of CoA-derivatives. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that of the wild-type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl-CoA-binding residues with hydrophobic amino acids caused a ligand-induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate-mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant-catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl-CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl-CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.