Active Site Heterogeneity Research Articles

Recent Advances on Computational Modeling of Supported Single-Atom and Cluster Catalysts: Characterization, Catalyst–Support Interaction, and Active Site Heterogeneity

To satisfy the need for catalyst materials with high activity, selectivity, and stability for energy conversion, material design and discovery guided by theoretical insights are a necessity. In the past decades, the rise in theoretical investigations into the properties of catalyst materials, reaction mechanisms, and catalyst design principles has shed light on the catalysis field. Quantitative structure–activity relationships have been developed through incorporating spectroscopic simulations, electronic structure calculations, and reaction mechanistic studies. In this review, we report the state-of-the-art computational approaches to catalyst materials characterization for supported single-atom and cluster catalysts utilizing spectroscopic simulations, i.e., XANES simulation, and material properties investigation via electronic-structure calculations. Furthermore, approaches regarding reaction mechanisms, focusing on active site heterogeneity, are also discussed.

Beyond upper bound estimates of active site densities in heterogeneous catalysis: A note on the critical role of titrant pressure

In-situ titrations employed in the estimation of active site densities oftentimes only provide upper bound estimates, not true active site densities. Discounting unselective contributions that result in a disparity between the two requires a treatment of transient titration data more nuanced than merely integrating under the titrant breakthrough curve. Three methods for estimating site densities (and turnover frequencies) are presented in the context of ceria-catalyzed ethene hydrogenation, and their strengths and limitations discussed. Readily accessible supra-monolayer coverages of CO prevalent on ceria surfaces help bring into relief considerations relating to unselective adsorption and active site heterogeneity common, yet infrequently addressed, in the characterization of heterogeneous catalysts using in-situ titration methods.

Computational Investigation of the Role of Active Site Heterogeneity for a Supported Organovanadium(III) Hydrogenation Catalyst

Computational Investigation of the Role of Active Site Heterogeneity for a Supported Organovanadium(III) Hydrogenation Catalyst

Active-Site Heterogeneity of Lactate Dehydrogenase

Molecular dynamics simulation of human heart lactate dehydrogenase (LDH) has been carried out to determine the linear and two-dimensional Fourier transform infrared (2D-FTIR) spectra for the carbonyl stretch vibration of pyruvate in the tetrameric enzyme, using quantum vibrational perturbation theory. The computed line-shapes of individual subunits are inhomogeneously broadened and span the entire absorption range of the carbonyl vibration of the full enzyme, indicating the similar conformation heterogeneity in the four active sites of LDH. However, each subunit line-shape has different width and peak maximum due to variations in conformation equilibrium in different subunits, corresponding to the spectral multiplets observed experimentally. Since there is a finite time interval before a substrate is converted into products in a given active site, the distribution of such a time coarse-grained average of Michaelis complexes is called active-site heterogeneity. Active-site heterogeneity is distinguished fr...

Strain effect and dual initiation pathway in CrIII/SiO2 polymerization catalysts from amorphous periodic models

Heterolytic CH bond activation of ethylene on CrIIIO has been proposed as the initial step in olefin polymerization on (SiO)3CrIII active sites of molecularly-defined analogues of the Phillips catalyst. Here, by using realistic amorphous periodic models that account for structural complexity, strain, and active site heterogeneity of well-defined silica-supported CrIII-based polymerization catalysts, we show that this activation step is significantly favored due to the strain present on highly dehydroxylated silica. Furthermore, we find that initiation by insertion of ethylene into the CrO bond is even more favorable, especially for more strained sites, while both mechanisms can compete for less strained and thereby less active sites. Our results suggest a competing dual pathway for ethylene polymerization on CrIII sites and are consistent with a distribution of active sites, the experimentally observed broad distribution of polymer molecular weight, and the increased polymerization activity upon high-temperature calcination in Phillips catalysts.

Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb–Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb–Hp exhibited active-site heterogeneity and noncooperative, high-affinity O2 binding (n1/2=0.88, P1/2=0.33mm Hg in inorganic phosphate buffer at pH 7 and 25°C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O2 dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb–Hp had significantly reduced absorption at the λmax of 430nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E1/2) of Hb–Hp was found to be +54mV, showing that it is much more easily oxidized than uncomplexed Hb (E1/2=+125mV). The Nernst plots for Hb–Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb–Hp was unchanged by pH over the range of 6.4–8.3. Exposure of Hb–Hp to excess hydrogen peroxide (H2O2) produced ferryl heme, which was found to be more kinetically inert in the Hb–Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb–Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb–Hp complex.

Olefin polymerization on immobilized zirconocene catalysts containing alkylaluminoxanes synthesized on the support surface

Ethylene and propylene polymerization on immobilized catalysts of composition MMT-H2O/AlR3/Zr-cene (MMT = montmorillonite) and on the corresponding homogeneous catalysts of composition Zr-cene-MAO (Zr-cene = rac-Et(Ind)2ZrCl2, rac-Me2Si(Ind)2ZrCl2, rac-Me2Si(2-Me-4-Ph-Ind)2ZrCl2, rac-1-(9-η5-Flu)2-(5,6-Cp2-Me-1-η5-Ind)Et]ZrCl2) is considered. Here, the activating support for the zirconocenes is montmorillonite containing methylaluminoxane or isobutylaluminoxane synthesized directly on the monmorillonite surface by the partial hydrolysis of an alkylaluminum (AlMe3, Al(i-Bu)3, Al(i-Bu)2H) with the mobile water of the support (MMT-H2O/AlR3). The MMT-H2O/AlR3 supports are demonstrated to be effective activators for ansa-zirconocenes. The catalytic properties of the immobilized systems (process kinetics and efficiency, the molar mass of the resulting polymer, and the structure of the macromolecules) depend on the activating support and the zirconocene precatalyst. The complexes of the MMT-H2O/Al(i-Bu)3 support with all zirconocene precatalysts are more active in propylene polymerization than the same complexes of MMT-H2O/AlMe3. The zirconcenes immobilized on MMT-H2O/AlR3 afford polyethylene and polypropylene with a higher molar mass than the corresponding homogeneous systems. Furthermore, immobilization causes activesite heterogeneity. As compared to the homogeneous single-site catalysts of composition Zr-cene-MAO, the corresponding catalysts immobilized on MMT-H2O/AlR3/Zr-cene are more stereospecific in the case of rac-Me2Si(Ind)2ZrCl2 (C2 symmetry) and are less stereospecific in the case of rac-[1-(9-η5-Flu)2-(5,6-Cp-2-Me-1-η5-Ind)Et]ZrCl2 (Cs symmetry).

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy.

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.

N-Butane isomerization on sulfated zirconia: active site heterogeneity and deactivation

The fast deactivation of sulfated zirconia (SZ) has limited its use in commercial processes such as n-butane isomerization. In order to investigate this deactivation, steady-state isotopic transient kinetic analysis (SSITKA) was utilized to study in situ changes in surface kinetic parameters for n-butane isomerization on a widely studied SZ at 150°C. Approximately 20% of the sulfate species was found to be n-butane adsorption sites, but only 1–2% of the sulfate species appeared to adsorb active surface reaction intermediates. The decrease in catalytic activity during deactivation could be attributed to the loss of active sites. The change in TOF ITK ∗ (TOF based on an average residence time of active surface intermediates) and the regeneration characteristics of the SZ catalyst suggest a possible active site heterogeneity. It appears that the high initial activity and the fast deactivation for TOS≤100 min were mainly due to the presence and deactivation of the more active sites, respectively. Following the loss of the more active sites, the less active sites provided the majority of the catalytic activity observed for TOS≥100 min . The less active sites appeared to be more easily regenerated than the more active sites as the catalytic activity at TOS≥100 min was recovered following regeneration at 315°C. Loss of active sites due to sulfur loss or migration seems unlikely. Site blockage by coke/oligomer formation appeared to be a significant contributor for catalyst deactivation for n-butane isomerization on SZ. The impact of sulfur reduction on catalyst deactivation cannot be ruled out at this point.

Spectroelectrochemistry of heme proteins: effects of active-site heterogeneity on Nernst plots

In order to detect and model the effect of functional chain heterogeneity on Nernst plots for heme proteins, we examined the redox properties of various myoglobins (Mbs) and their mixtures using an improved spectroelectrochemical method. Specific redox responses and formal half potentials ( E 1/2) were obtained for Aplysia, horse, and sperm whale Mbs, as well as 1:1 mixtures of Mbs consisting of Aplysia/sperm whale, sperm whale/horse, and horse/ Aplysia. Linear Nernst plots with slopes near unity were observed for horse, sperm whale, and Aplysia Mbs, with E 1/2 values of 14, 19, and 96 mV (vs. NHE) respectively, consistent with previous reports using indirect methods. The Nernst plot responses for mixtures of some of these Mbs allowed us to evaluate and model the non-Nernstian behavior that results from intrinsically different values of E 1/2 and from incomplete spectral overlap. The data demonstrate that increasing the E 1/2 differences between the components of a Mb mixture increases the changes in shape of the resulting Nernst plots, the dominant effect being a decrease in the observed Nernst coefficient ( n Nernst). Comparison of Nernst plots for redox data with Hill plots for O 2 binding data shows that the redox process is more affected by the structural differences in the distal heme pockets of the Mbs studied than is O 2 binding. Similar effects of chain heterogeneity may give rise to disproportionate reductions in the slopes of Nernst and Hill plots for hemoglobins (Hbs). This possibility is discussed in relation to Hbs investigated for redox and O 2 binding activity in our laboratories where we find n Nernst to be commonly less than n Hill over a range of experimental conditions.

An EPR investigation on the structural heterogeneity in copper azurin and plastocyanin

The effects of cooling rate and of solvent properties on the active site heterogeneity of two copper proteins, azurin and plastocyanin, have been investigated at low temperature by electron paramagnetic resonance spectroscopy. The spectra of theses proteins have been analyzed, by an accurate computer simulation, in terms of a distribution of some relevant spin-Hamiltonian parameters. The results show that the structural heterogeneity of both proteins, quantified by the width of the distribution in the g and A tensors, is affected by both the freezing procedure and the solvent composition. In particular, the g distribution width is found to be reduced in the slow cooling regime; such a reduction appearing more significant when glycerol is added to the protein solutions. Despite of the similarity in the copper ion microenvironments of the two proteins, the effects are more pronounced in azurin. The results are discussed also in connection with the role played by the solvent and the rate of freezing in featuring the conformational substate landscape.

Active-site heterogeneity in Limulus hemocyanin as revealed by reaction with peroxides

Previously reported differences in the reactivities toward active-site ligands such as hydrogen peroxide indicate that the active-site geometries of the arthropod and mollusc hemocyanins are significantly different. Results are presented which demonstrate that the purified subunits composing the native hemocyanin of an arthropod show comparable active-site heterogeneity. Neither whole nor stripped samples of Limulus oxyhemocyanin are completely oxidized by reaction with hydrogen peroxide. Moreover, the addition of hydrogen peroxide to deoxy or completely oxidized samples causes partial regeneration to the oxy form. This behavior is most pronounced for one of the five major chromatographic fractions, Zone III, which retains greater than 60% of its copper-oxygen absorbance band when treated with hydrogen peroxide. Zone III is composed of three subunits (IIIA, IIIB, and IIIB′). With excess hydrogen peroxide, the deoxy and oxidized forms of Subunit IIIA can be completely regenerated to a fully functional oxy state, while Subunits IIIB and IIIB′ are less than 30% regenerated. Upon long-term storage, Subunit IIIA slowly loses 340 nm absorbance, an aging process that can be reversed by hydrogen peroxide. These features of Subunit IIIA, that set it apart from other subunits of the 48-member ensemble of intact Limulus hemocyanin, are typically exhibited by mollusc hemocyanins rather than arthropod hemocyanins. These findings clearly illustrate that significantly different active-site geometries can occur within a single type of metalloprotein and suggest that comparisons between the various Limulus subunits and Subunit IIIA may prove valuable in unraveling the structural basis for the disparity in the chemical reactivity exhibited by hemocyanins of different species.

Separation of SH-modified myosin subfragment-1 (A1) isozyme into two distinct equimolar fractions by an affinity chromatography.

Our previous kinetic studies indicated that SH-modified myosin subfragment-1 A1 isozyme (S1(A1] contains at least two different types of active sites (Emoto, Y., Kawamura, T., & Tawada, K. (1985) J. Biochem. 98, 735-745). In those studies we have modified highly reactive SH-groups in S1(A1) with thimerosal. In this work, we separated the modified S1(A1) into two equimolar fractions by affinity chromatography with agarose-ADP. For the separation, Mg2+ in the elution buffer was indispensable. Although the two fractions appeared to have the same number of modified SH-groups per mol of S1, they had different enzymic and fluorescent properties. SH-modification with an excess of thimerosal for a much longer duration did not change any of the results: not the chromatographic profile, the properties of the two fractions, nor the number of modified SH-groups. Hence the two different populations were not generated by incomplete modification. After reduction with dithiothreitol, however, the differences between the two fractions disappeared. When we separately re-modified the reduced fractions and re-chromatographed them, in each case we again obtained two fractions, which had the same properties as the two fractions obtained from the original modification with thimerosal. These results demonstrate that the active site heterogeneity in SH-modified S1(A1) had no intrinsic origin in the unmodified S1: it was introduced by the SH-modification, but by an unknown mechanism(s) other than incomplete modification.

Characterization of the ATPase active site in myosin subfragment-1 with the use of vanadate plus ADP as a reversible "affinity-labeling" reagent: evidence for heterogeneity in the active sites.

Our previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by "affinity-labeling" analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S1) and one of the isozymes, S1(A1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S1 or S1(A1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.

The specific activity value based on the prosthetic copper of ascorbate oxidase

The copper content and activity data of 137 purified samples of ascorbate oxidase ( l-ascorbate:O 2 oxidoreductase, EC 1.10.3.3) prepared in this laboratory during the period 1951–1977 have been examined and correlated. These data support the developing concepts of “active site heterogeneity” in otherwise homogeneous protein preparations. The specific activities, based on the copper contents of these 137 enzyme specimens, have been determined to average at about 760 units per μg copper and to reach maximum values in the area of 1000 units per μg copper. The maximum specific activity value (units per mg protein) and the copper content value of these purified specimens have been found to be 3800 ± 400 and 0.46 ± 0.06%, respectively.