Properties Of Active Site Research Articles

ChemInform Abstract: Variable Temperature IR Spectroscopy in the Studies of Oxide Catalysts

AbstractReview: 25 refs.

Investigation on Process Mechanism on Cu–Cr Catalysts for Ethanol Dehydrogenation to Ethyl Acetate

The Cu–Cr catalysts were prepared by co-precipitation method,and the role of active sites on Cu–Cr catalyst for direct synthesis of ethyl acetate from ethanol was investigated. The catalysts were characterized by means of XRD, TEM, XPS, ethanol-TPSR, EA-TPD and FT-IR etc., respectively. The results show that the outermost surface layer of Cu–Cr catalysts after reduction is composed of Cu0, Cr2O3 and CuCr2O4 phases. The ethanol dehydrogenation step is carried out on Cu0 active sites, but the selectivity of ethyl acetate is not remarkably influenced by the properties of Cu0 active sites, the proper performances of Lewis acidic sites on the Cu–Cr catalyst might play a key role to improve the selectivity of ethyl acetate.

ChemInform Abstract: Molybdenum and Tungsten Oxidoreductase Models

AbstractReview: about 50 refs.

Time-Resolved and Steady-State Spectroscopy of Native and Mutated Thermobifida Fusca Hemoglobins

Thermobifida fusca hemoglobin (TFH) is a prototypical bacterial (class 2) hemoglobin. It has been identified in a thermophilic actinobacterium and overexpressed. The heme cavity properties are mainly related to the polarity and H-bonding capability of the “distal” amino acids: tyrosine (B10 in the myoglobin helix notation), tyrosine (CD1), tryptophan (G8). Single, double and triple phenyalalanin (Phe) mutants of these key residues have recently become available. We have applied several techniques of time-resolved and steady-state optical spectroscopy to the study of THF carbon monoxide complexes, in an effort to correlate structural and dynamic properties of the protein active site. i) Femtosecond transient absorption has shown that CO can rebind from TFH distal cavity in few ns. TrpG8 substitution with the apolar Phe accelerates the recombination, which takes place in hundreds of ps. ii) Laser flash photolysis has allowed to extend the study of the dynamics to longer times. The yield of the geminate rebinding and the kinetics of recombination from the solvent are both influenced by the single TrpG8 → Phe mutation. Further changes are observed when the three polar amino acids are replaced by Phe. Rate constant distributions have been obtained by the maximum entropy method. iii) Photoacoustic measurements have yielded both volume and enthalpy changes following CO photodissociation in the ns range. The thermodynamic parameters can be related to those measured by laser flash photolysis. iv) Resonance Raman spectroscopy of seven THF mutated variants has given evidence for a H-bonding network in the distal cavity, which involves the bound CO. All the results point to an overwhelming role for TrpG8. This is in agreement with a recent study on a related bacterial hemoglobin from B. subtilis.

Understanding Functional Tuning, Robustness, and Evolvability in Proteins By High-Throughput Biophysics

Proteins consist of only 20 different amino acids with very modest chemical reactivity, but perform a breathtaking range of functions. A central question in biophysics is to understand how proteins achieve such functional versatility. A key recent advance in understanding protein structure-function relationships is the robustness of proteins against point mutations, which implies that only a small subset of residues determines functional properties. We tested this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PAS domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. Our high-throughput biophysical measurements on a complete ala scan of purified PYP mutants characterizing active site properties, functional kinetics, stability, and production level reveal that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties (Philip et al., PNAS 2010, early edition 10.1073/1006660107). Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. The photocycle kinetics are significantly altered by substitutions at 58 positions and span a 3,000-fold range, allowing us to identify conserved interactions governing allosteric switching in PYP. The data also shed light on two classic examples of functional tuning of active site groups, shifts in pKa value and shifts in absorbance maximum for spectral tuning, and result in a novel model for spectral tuning based on changes in the shape of the energy surfaces involved (Philip et al., 2010, PNAS 107: 5821-5826). The results show that PYP combines robustness with a high degree of evolvability, imply production level as an important factor in protein evolution, and provide new insights into the functional tuning of active site residues.

Methane oxidation on Pd–Ceria: A DFT study of the mechanism over Pd xCe 1−xO 2, Pd, and PdO

Palladium/ceria exhibits unique catalytic activity for hydrocarbon oxidation; however, the chemical and structural properties of active sites on the palladium–ceria surface are difficult to characterize. Strong interactions between palladium and the ceria support stabilize oxidized Pd δ + species, which may contribute to the significant activity of Pd/ceria for methane oxidation. We present a density functional theory (DFT + U) investigation into methane oxidation over Pd/ceria and quantify the activity of the Pd x Ce 1− x O 2(1 1 1) mixed oxide surface in comparison with the PdO(1 0 0) and Pd(1 1 1) surfaces. The methane activation barrier is lowest over the Pd x Ce 1− x O 2(1 1 1) surface, even lower than over the Pd(1 1 1) surface or low coordinated stepped or kinked Pd sites. Subsequent reaction steps in complete oxidation, including product desorption and vacancy refilling, are considered to substantiate that methane activation remains the rate-limiting step despite the low barrier over Pd x Ce 1− x O 2(1 1 1). The low barrier over the Pd x Ce 1− x O 2(1 1 1) surface demonstrates that mixed ceria-noble metal oxides offer the potential for improved hydrocarbon oxidation performance with respect to dispersed noble metal particles on ceria.

Theoretical studies on electronic structure and magnetic properties of mixed-valence uteroferrin active site

The electronic structure and magnetic interaction of the active site of pig purple acid phosphatase (PAP, uteroferrin) were investigated using pure DFT (UBLYP) and hybrid DFT methods (UB3LYP and UB2LYP). Uteroferrin catalyzes the hydrolysis of a phosphate ester under acidic conditions and contains a binuclear iron center. The mammalian PAPs are expected to be targets for drug design of osteoporosis. Their active sites are typical examples of the Fe(II)-Fe(III) mixed-valence system. We studied double exchange interaction of the mixed-valence system, using the potential energy difference between the Fe(II)-Fe(III) and the Fe(III)-Fe(II) states. The pathway of the antiferromagnetic coupling between Fe(III) and Fe(II) were also discussed by using chemical indices, which are evaluated by the occupation numbers of singly occupied natural orbitals. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Metal-Binding Loop Length Is a Determinant of the pKa of a Histidine Ligand at a Type 1 Copper Site

The type 1 copper site of a cupredoxin involves coordination by cysteine, histidine, and methionine residues from a single loop. Dissociation and protonation of the histidine ligand on this loop is observed in only certain reduced cupredoxins and can regulate electron-transfer reactivity. This effect is introduced in azurin (AZ) (the wild-type protein has an estimated pKa of <2) by mutating the native copper-binding loop (C(112)TFPGH(117)SALM(121), ligands numbered). In this work, we have investigated the influence of loop length alone on histidine ligand protonation by determining the pKa value in AZ variants with ligand-containing polyalanine loops of different length. Crystal structures of the Cu(I)-variant with the loop sequence C(112)AAH(115)AAM(118) (AZ2A2A) demonstrate that at pH 4.2 His115 is protonated and no longer coordinated, and the imidazole ring is rotated by 180°. The influence of pH on the reduction potential allows a pKa of 5.2 ± 0.1 for His115 in Cu(I)-AZ2A2A to be determined. In the reduced AZ variants in which the loop sequences C(112)AAAAH(117)AAAM(121) (AZ4A3A) and C(112)AAAAH(117)AAAAM(122) (AZ4A4A) have been introduced, pKa values of 4.5 ± 0.1 and 4.4 ± 0.1, respectively, are obtained for the His117 ligand. Consistent with these data, the crystal structure of Cu(I)-AZ4A4A at pH 5.3 shows no sign of His117 protonation (crystals were unstable at lower pH values). The loop length range studied matches that which occurs naturally and these investigations indicate that length alone can alter the pKa of the coordinating histidine by approximately 1 pH unit. The pKa for this histidine ligand varies in native cupredoxins by >5 pH units. Other structural and electronic features, governed primarily by the second-coordination sphere, to which the ligand-binding loop is a major contributor, also alter this important feature. A longer ligand-containing loop made of residues whose side chains are larger and more complex than a methyl group increases the second coordination sphere providing additional scope for tuning the pKa of the histidine ligand and other active site properties.

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.

Crystal Structures and Mutational Analyses of Acyl-CoA Carboxylase β Subunit of Streptomyces coelicolor,

The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, E. coli

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T (2), of the dinuclear coupled pair Fe(III)-Cu(B)(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(B)(II) coupled by approximately 1.5-3 cm(-1) to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.

Protein–Protein Complex Formation Affects the Ni–Fe and Fe–S Centers in the H2‐Sensing Regulatory Hydrogenase from Ralstonia eutropha H16

The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ.

High‐Field2H‐Mims‐ENDOR Spectroscopy on PSII Single Crystals: Hydrogen Bonding of YD.

Hydrogen bonds are key determinants for protein structures and the fine-tuning of active-site properties. For example, they are responsible for the redox potential variability of protein-imbedded chromophores. By applying high-field (94 GHz) Mims-ENDOR spectroscopy on deuterium-exchanged frozen-solution samples and single crystals of photosystem II from Th. elongatus, we identified the hydrogen-bonding partner of the tyrosyl radical D2-Tyr160, Y(D)*, directly by the strength and orientation of the deuterium hyperfine coupling as D2-His189, that is, without relying on the disappearance of a hyperfine coupling interaction upon deletion of D2-His189. No indications for additional hydrogen bonds can be found in the spectra, thereby eliminating hypotheses about a water network as hydrogen-binding partner of Y(D)*.

Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, K(M) values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging.

Electron spin resonance study of Mo(V) ion species incorporated into aluminosilicate nanospheres with solid core/mesoporous shell structure

Silica spheres with sub-micrometer sized solid core and mesoporous shell (SCMS) structure were synthesized, and aluminum was incorporated into the mesoporous shell framework by impregnation method to generate SCMS aluminosilicate (AlSCMS) nanospheres. The impregnation of aluminum into the SCMS spheres generates the acid sites on the framework due to the presence of Al3+ ions. The AlSCMS was then used to support molybdenum ion species on the mesoporous shell framework. A solid-state reaction of MoO3 with AlSCMS followed by thermal reduction generated paramagnetic Mo(V) species. The dehydration produced a Mo(V) species that is characterized by electron spin resonance with ge > g⊥ > g||. The structural properties of active sites in the AlSCMS were characterized by means of XRD, UV–Vis, 27Al MAS NMR, FT-IR, and energy dispersive X-ray spectrometric measurements. Upon O2 adsorption, the Mo(V) ESR signal intensity decreased, and a new O2− radical was generated. The Mo species in the dehydrated Mo-AlSCMS is found to exist as oxo-molybdenum species, (MoO2)+ or (MoO)3+. Since the AlSCMS has a low framework negative charge, the MoO2+ with a low positive charge can be easily stabilized and thus seems to be more probable in the AlSCMS framework.

Evaluation of electromagnetic enhancement of surface enhanced hyper Raman scattering using plasmonic properties of binary active sites in single Ag nanoaggregates

We found a binary active site of surface enhanced Raman scattering (SERS) and surface enhanced hyper Raman scattering (SEHRS) in single Ag nanoaggregates by single particle spectroscopy. The intensity fluctuation of SEHRS was canceled by dividing SEHRS intensity by SERS intensity on the basis of the binary active site analysis. Thanks to the identification of the plasmons common to both SERS and SEHRS we revealed that an enormous enhancement in SEHRS is ascribed to the electromagnetic (EM) enhancement entirely coupled with longitudinal plasmon modes excluding other kinds of enhancement factors. Our results indicate that EM enhancement factors of metal nanostructures are estimated from spectral information on the longitudinal plasmon resonance band obtained by the scattering or absorption spectra of the nanostructures.

Nickel(II)-substituted azurin I from Alcaligenes xylosoxidans as characterized by resonance Raman spectroscopy at cryogenic temperature

Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal-ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the nu(Cu-S)(Cys) markers at 373, 399, 409, and 430 cm(-1). Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S --> M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal-thiolate bonding interaction, indicated by substantially lower nu(Ni-S)(Cys) stretching frequencies, (3) elevation of the cysteine nu(C( beta )-S) stretching, amide III, and rho (s)(C( beta )H(2)) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic nu(Ni-S)(Cys) stretching RR bands at 347, 364, and 391 cm(-1). Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)-S(Cys) stretching frequencies are calculated to be nu(Cu-S)(iwa) = 406.3 and 407.6 cm(-1), respectively. These values decreased to nu(Ni-S)(iwa) = 359.3 and 365.5 cm(-1), respectively, after Ni(II) --> Cu(II) exchange, suggesting that the metal-thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.

Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the Transmembrane Oxidoreductase DsbD

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys103-Cys109 disulfide of nDsbD by Cys461 of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol.370 ,643 -65817544440) that the attacking cysteine (Cys461) in isolated cDsbD has a high pKa value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pKa values change significantly when cDsbD forms a complex with nDsbD. This modulation of pKa values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pKa values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.

π-Interaction Tuning of the Active Site Properties of Metalloproteins

The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.

Role of the variable active site residues in the function of thioredoxin family oxidoreductases

The enzymes of the thioredoxin family fulfill a wide range of physiological functions. Although they possess a similar CXYC active site motif, with identical environment and stereochemical properties, the redox potential and pK(a) of the cysteine pair varies widely across the family. As a consequence, each family member promotes oxidation or reduction reactions, or even isomerization reactions. The analysis of the three-dimensional structures gives no clues to identify the molecular source for the different active site properties. Therefore, we carried out a set of quantum mechanical calculations in active site models to gain more understanding on the elusive molecular-level origin of the differentiation of the properties across the family. The obtained results, together with earlier quantum mechanical calculations performed in our laboratories, gave rise to a consistent line of evidence, which points to the fact that both active site cysteines play an important role in the differentiation. In contrary to what was assumed, differentiation is not achieved through a different stabilization of the solvent exposed cysteine but, instead, through a fine tuning of the nucleophilicity of both active site cysteines. Reductant enzymes have both cysteine thiolates poorly stabilized, oxidant proteins have both cysteine thiolates highly stabilized, and isomerases have one thiolate (solvent exposed) poorly stabilized and the other (buried) thiolate highly stabilized. The feasibility of shifting the chemical equilibrium toward oxidation, reduction, or isomerization only through subtle electrostatic effects is quite unusual, and it relies on the inherent thermoneutrality of the catalytic steps carried out by a set of chemically equivalent entities all of which are cysteine thiolates. Such pattern of stabilization/destabilization, detected in our calculations is fully consistent with the observed physiological roles of this family of enzymes.