Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions.

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.     

Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus.

NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at approximately 70 degrees C for T. thermophilus. At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0-1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 degrees C. At higher urea concentrations the enzyme activity is inhibited. The urea-dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, deltaH* and TdeltaS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.     

Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids

The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water.     

Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54?kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

Hofmeister effects on activity and stability of alkaline phosphatase

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients (B₊ for cations and B₋ for anions). The catalytic activity or V_max/K_m of the enzyme showed a bell-shaped relationship with the (B₋ − B₊) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO₄²⁻ (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.     

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.     

Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli.

The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli-borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.     

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c

Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80–heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80–heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.     

Studies of a halophilic NADH dehydrogenase. II. Kinetic properties of the enzyme in relation to salt activation.

1. An NADH dehydrogenase, obtained from an extremely halophilic bacterium, was activated by various salts when enzyme activity was measured as the observed velocity, whereas the maximum velocity was unaffected by either the salt concentration or the nature of the salt. 2. Two ion effects were observed; a quantitative cation effect, reflected in changes in the apparent Michaelis constant for 2,6-dichlorophenolindophenol, and a qualitative anion effect, reflected in the apparent Michaelis and dissociation constants for NADH. 3. The data suggest that cations act by neutralizing electrostatic charges surrounding the 2,6-dichlorophenolindophenol-binding site, whereas the anions affect the conformation of the enzyme by altering the accessibility of the NADH-binding site to the bulk solvent. 4. Thus, the apparent activation of this enzyme, obtained from an extremely halophilic bacterium, is a reflection of measuring enzyme activity at non-saturating substrate concentrations.     

An investigation into the protective effect of various salts on the acid inactivation of human serum butyrylcholinesterase (EC 3.1.1.8)

Human serum butyrylcholinesterase (EC 3.1.1.8) loses 100% of its activity toward butyrylthiocholine in 60 min atpH 3.0. This deactivation is retarded by 1.37 M ammonium sulfate to a loss of 40% after 60 min atpH 3.0. Reneutralization experiments suggest that the mechanism for this acid inactivation does not exclusively involve hydrolysis of peptide bonds or protonation of the enzyme's active site. Studies with different anions and cations demonstrate that the order of their effectiveness as protective agent against acid inactivation closely follows the Hofmeister series. No relationship was found between catalytic activation or inhibition by salt and protection from acid inactivation. Ultraviolet difference studies at 288 nm with enzyme brought topH 2.7 frompH 8.0 in the presence and absence of 1.37 M ammonium sulfate demonstrated no change in UV absorbance with ammonium sulfate present and approximately a 0.15 ODU rise in absorbance in the absence of ammonium sulfate. These results suggest that acidicpH conditions result in deactivating stereochemical changes in the active site of butyrylcholinesterase and that certain anions and cations, according to the Hofmeister series, are able to protect the enzyme from acid inactivation by stabilizing the active conformation of its active site.     

Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR.     

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)⁺ with the concurrent reduction of O₂ to H₂O₂, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and ¹⁵N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.     

Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions

This paper examined the effect of several pyridinium and imidazolium-based ionic liquids (ILs) on the protease stability in aqueous solutions. In general, the enzyme was found quite active at low concentrations of hydrophilic ILs. In aqueous environment, the enzyme was stabilized by the kosmotropic anions (such as CF3COO- and CH3COO-) and chaotropic cations (such as [BuPy]+ and [EMIM]+), but was destabilized by chaotropic anions (such as tosylate and BF4-) and kosmotropic cations (such as [BMIM]+).     

Ion-induced alterations of the local hydration environment elucidate Hofmeister effect in a simple classical model of Trp-cage miniprotein

Protein stability is known to be influenced by the presence of Hofmeister active ions in the solution. In addition to direct ion-protein interactions, this influence manifests through the local alterations of the interfacial water structure induced by the anions and cations present in this region. In our earlier works it was pointed out that the effects of Hofmeister active salts on the stability of Trp-cage miniprotein can be modeled qualitatively using non-polarizable force fields. These simulations reproduced the structure-stabilization and structure-destabilization effects of selected kosmotropic and chaotropic salts, respectively. In the present study we use the same model system to elucidate atomic processes behind the chaotropic destabilization and kosmotropic stabilization of the miniprotein. We focus on changes of the local hydration environment of the miniprotein upon addition of NaClO₄ and NaF salts to the solution. The process is separated into two parts. In the first, ‘promotion’ phase, the protein structure is fixed, and the local hydration properties induced by the simultaneous presence of protein and ions are investigated, with a special focus on the interaction of Hofmeister active anions with the charged and polar sites. In the second, ‘rearrangement’ phase we follow changes of the hydration of ions and the protein, accompanying the conformational relaxation of the protein. We identify significant factors of an enthalpic and entropic nature behind the ion-induced free energy changes of the protein-water system, and also propose a possible atomic mechanism consistent with the Collins’s rule, for the chaotropic destabilization and kosmotropic stabilization of protein conformation.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Conformational stability and dynamics of cytochrome <Emphasis Type="Italic">c</Emphasis> affect its alkaline isomerization

The alkaline isomerization of horse heart ferricytochrome c (cyt c) has been studied by electronic absorption spectroscopy in the presence of the Hofmeister series of anions: chloride, bromide, rhodanide and perchlorate. The anions significantly affect the apparent pK _a value of the transition in a concentration-dependent manner according to their position in the Hofmeister series. The Soret region of the absorption spectra is not affected by the presence of the salts and shows no significant structural perturbation of the heme crevice. In the presence of perchlorate and rhodanide anions, the cyanide exchange rate between the bulk solvent and the binding site is increased. These results imply higher flexibility of the protein structure in the presence of chaotropic salts. The thermal and isothermal denaturations monitored by differential scanning calorimetry and circular dichroism, respectively, showed a decrease in the conformational stability of cyt c in the presence of the chaotropic salts. A positive correlation between the stability, ΔG, of cyt c and the apparent pK _a values that characterize the alkaline transition indicates the presence of a thermodynamic linkage between these conformational transitions. In addition, the rate constant of the cyanide binding and the partial molar entropies of anions negatively correlate with the pK _a values. This indicates the important role of anion-induced solvent reorganization on the structural flexibility of cyt c in the alkaline transitions. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Anion and cation effects on the structural stability of lysozyme were investigated using differential scanning calorimetry. At low concentrations (<5 mM) anions and cations alter the stability of lysozyme but they do not follow the Hofmeister (or inverse Hofmeister) series. At higher concentrations protein stabilization follows the well‐established Hofmeister series. Our hypothesis is that there are three mechanisms at work. At low concentrations the anions interact with charged side chains where the presence of the ion can alter the structural stability of the protein. At higher concentrations the low charge density anions perchlorate and iodide interact weakly with the protein. Their presence however reduces the Gibbs free energy required to hydrate the core of the protein that is exposed during unfolding therefore destabilizing the structure. At higher concentrations the high charge density anions phosphate and sulfate compete for water with the protein as it unfolds increasing the Gibbs free energy required to hydrate the newly exposed core of the protein therefore stabilizing the structure.     

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.