Structural and binding studies of C-terminal half (C-lobe) of lactoferrin protein with COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs)

Three COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs), etoricoxib, parecoxib, and nimesulide are widely prescribed against inflammatory conditions. However, their long term administration leads to severe conditions of cardiovascular complications and gastric ulceration. In order to minimize these side effects, C-terminal half (C-lobe) of colostrum protein lactoferrin has been indicated to be useful if co-administered with NSAIDs. Lactoferrin is an 80 kDa glycoprotein with two similar halves designated as N- and C-lobes. Since NSAID-binding site is located in the C-terminal half of lactoferrin, C-lobe was prepared from lactoferrin by limited proteolysis using proteinase K. The incubation of lactoferrin with serine proteases for extended periods showed that N-lobe was completely digested but C-lobe was resistant for more than 72 h indicating its long half life in the animal gut. The solution studies have shown that COX-2-specific NSAIDs bind to C-lobe with binding constants ranging from 10⁻⁴ to 10⁻⁵ M showing significant affinities for sequestering these compounds. In order to understand the mode of binding and sequestering properties, the complexes of C-lobe with all these three compounds, etoricoxib, parecoxib, and nimesulide were prepared and the structures of their complexes with C-lobe were determined at 2.2, 2.9, and 2.7 ? resolutions, respectively. The analysis of the structures of complexes of C-lobe with NSAIDs clearly show that all the three compounds bind firmly at the same ligand-binding site in the C-lobe revealing the details of the interactions between C-lobe and NSAIDs. The mode of binding of COX-2-specific NSAIDs to C-lobe is similar to that of the binding of COX-2 non-specific NSAIDs to C-lobe.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Binding of ruthenium(III) anti-tumor drugs to human lactoferrin probed by high resolution X-ray crystallographic structure analyses

The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm₂Cl₄], HInd[RuInd₂Cl₄] and (HInd)₂ [RuIndCl₅], where Im?=?imidazole and Ind?=?indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6?Å, 2.2?Å and 2.4?Å respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3?Å data) and 0.192 (for 8.0 to 2.4?Å data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins.     

Some insights into the binding mechanism of the GABAA receptor: a combined docking and MM-GBSA study

Gamma-aminobutyric type A receptor (GABA_AR) is a member of the Cys-loop family of pentameric ligand gated ion channels (pLGICs). It has been identified as a key target for many clinical drugs. In the present study, we construct the structure of human 2α₁2β₂γ₂ GABA_AR using a homology modeling method. The structures of ten benzodiazepine type drugs and two non-benzodiazepine type drugs were then docked into the potential benzodiazepine binding site on the GABA_AR. By analyzing the docking results, the critical residues His102 (α₁), Phe77 (γ₂) and Phe100 (α₁) were identified in the binding site. To gain insight into the binding affinity, molecular dynamics (MD) simulations were performed for all the receptor–ligand complexes. We also examined single mutant GABA_AR (His102A) in complexes with the three drugs (flurazepam, eszopiclone and zolpidem) to elucidate receptor–ligand interactions. For each receptor–ligand complex (with flurazepam, eszopiclone and zolpidem), we calculated the average distance between the C_α of the mutant residue His102A (α₁) to the center of mass of the ligands. The results reveal that the distance between the C_α of the mutant residue His102A (α₁) to the center of flurazepam is larger than that between His102 (α₁) to flurazepam in the WT type complex. Molecular mechanic-generalized Born surface area (MM-GBSA)-based binding free energy calculations were performed. The binding free energy was decomposed into ligand-residue pairs to create a ligand-residue interaction spectrum. The predicted binding free energies correlated well (R ²?=?0.87) with the experimental binding free energies. Overall, the major interaction comes from a few groups around His102 (α₁), Phe77 (γ₂) and Phe100 (α₁). These groups of interaction consist of at least of 12 residues in total with a binding energy of more than 1 kcal mol^?1. The simulation study disclosed herein provides a meaningful insight into GABA_AR–ligand interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

α2-Macroglobulin-protease reactions: Relationship of covalent bond formation,methylamine reactivity,and specific proteolysis

Experiments were performed to define the relation between covalent binding of enzymes to β₂-macroglobulin (α₂M), the specific proteolysis of α₂M subunits to 85K fragments, and the reactivity of the methylamine site on α₂M. We studied the reaction of α₂M with native trypsin, anhydrotrypsin, and two active lysyl-blocked derivatives, methyl-trypsin and dimethylmaleyl-trypsin, the last with reversibly modified amino groups that can be regenerated at low pH. The results were: (1) All enzymes tested reacted with α₂M but only native trypsin formed covalent complexes (not dissociable by sodium dodecyl sulfate). Trypsin and the lysyl-blocked enzymes caused complete proteolysis of the α₂M subunits, in agreement with previous studies. (2) The dimethyl-maleyl-trypsin became covalently bound to α₂M only after removing the blocking groups of the bound enzyme, indicating that sequential proteolysis and covalent bond formation is possible. Under the conditions used for deblocking, there was no change in the covalent/noncovalent binding ratio of native trypsin, anhydrotrypsin, or the other lysyl-blocked derivative, methyl-trypsin. (3) Native trypsin or anhydrotrypsin displaced methyl- or dimethylmaleyl-trypsin from their α₂M complexes but the newly bound enzymes with free amino groups did not form covalent bonds indicating that enzymes must remain in association with the inhibitor for the bond to form. (4) Methylamine reacts with noncovalent α₂M complexes but not with covalent complexes. (5) Methylamine-treated α₂M can still form complexes with trypsin but at a drastically reduced rate and only noncovalent complexes are formed. In summary, sequential proteolysis and covalent bond formation is possible under certain conditions, and there is a strong correlation between covalent binding and loss of methylamine reactivity. The latter observation is suggestive evidence for the identity of the covalent binding site of α₂M and the putative thiol ester of the methylamine site. The enzyme lysyl amino groups, are likewise possible candidates for attacking nucleophile at that site.     

Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [³H]imidacloprid binding sites with different affinities (K_d = 3.5 ± 0.6 pM and 1.5 ± 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlα1, Nlα2 and Nlβ1 subunits into one receptor complex and of Nlα3, Nlα8 and Nlβ1 into another. Immunodepletion of Nlα1 or Nlα2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlα3 or Nlα8 caused the selective loss of the high-affinity site. Immunodepletion of Nlβ1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlα4, Nlα6, Nlα7 and Nlβ2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlα1, Nlα2 and Nlβ1 constitute the lower affinity binding site, whereas nAChRs containing Nlα3, Nlα8 and Nlβ1 constitute the higher affinity binding site for imidacloprid in N. lugens.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for α-globin over that for β-globin and intact HbA in an adenosine 5′-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the α-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in α-globin compared with intact haemoglobin, HbA and the β-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the α-globin chain over that of the β-globin in the haemoglobin disorder β-thalassaemia.     

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

Bovine lactoferrin promotes bifidobacterial growth. Its binding to bifidobacteria is thought to be responsible for such action. After separating the bovine lactoferrin half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding appeared when lactoferrin C-lobe was reacted with the cell surface proteins on a polyvinylidene difluoride membrane. Conversely, a 50-kDa band appeared when the surface proteins were reacted with either intact or nicked bovine lactoferrin. This result strongly suggests that the binding region could be lactoferrin N-lobe. Interestingly, despite the absence of binding, C-lobe enhanced bifidobacterial growth.     

The interaction of N-trifluoroacetylgalactosamine and its derivatives with winged bean (Psophocarpus tetragonolobus) basic agglutinin reveals differential mechanism of their recognition: a fluorine-19 nuclear magnetic resonance study

Here, we show the binding results of a leguminosae lectin, winged bean basic agglutinin (WBA I) to N-trifluoroacetylgalactosamine (NTFAGalN), methyl-α-N-trifluoroacetylgalactosamine (MeαNTFAGalN) and methyl-β-tifluoroacetylgalactosamine (MeβNTFAGalN) using ¹⁹?F NMR spectroscopy. No chemical shift difference between the free and bound states for NTFAGalN and MeβNTFAGalN, and 0.01-ppm chemical shift change for MeαNTFAGalN, demonstrate that the MeαNTFAGalN has a sufficiently long residence time on the protein binding site as compared to MeβNTFAGalN and the free anomers of NTFAGalN. The sugar anomers were found in slow exchange with the binding site of agglutinin. Consequently, we obtained their binding parameters to the protein using line shape analyses. Aforementioned analyses of the activation parameters for the interactions of these saccharides indicate that the binding of α and β anomers of NTFAGalN and MeαNTFAGalN is controlled enthalpically, while that of MeβNTFAGalN is controlled entropically. This asserts the sterically constrained nature of the interaction of the MeβNTFAGalN with WBA I. These studies thus highlight a significant role of the conformation of the monosaccharide ligands for their recognition by WBA I.     

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1-β1 and PFDα2-β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1-β1; CPNα and PFDα2-β2; CPNβ and PFDα1-β1; and CPNβ and PFDα2-β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNβ and PFDα2-β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2-β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1-β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2-β2 is specific for CPNβ.     

Binding and Cleavage of E. coli HUβ by the E. coli Lon Protease

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUβ, HUα, or HUβ-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUβ.     

Thermodynamics for base binding to four atropisomers cobalt(II) picket fence porphyrin. Intramolecular ligand–ligand interactions

Thermodynamic parameters for base binding to four atropisomers of meso-tetrakis(o-pivalamidophenyl)porphyrinatocobalt(II) were determined by spectrophotometry in toluene. The order of the affinities of the four isomers with 1-methylimidazole and pyridine is α⁴<α³<cis-α²<trans-α². The higher base affinities of the trans-α² complex compared with the α⁴ complex are due to an increase in the binding energies of the bases, although a substantial decrease of entropy changes also occurs; the differences of thermodynamic values on both complexes are −ΔΔG = 1.49 and 1.36 kcal/mol, −ΔΔH = 3.4 and 3.1 kcal/mol and −ΔΔS = 6.4 and 6.1 eu, with 1-methylimidazole and pyridine, respectively. With saturated bases pyrrolidine and piperidine, the affinities of the trans-α² complex are comparable to those of the α⁴ complex, and those of the cis-α² complex are the lowest. The increased steric repulsion between the pickets and ligated pyrrolidine or piperidine may cancel out the stabilizing effect on the base binding to the α² complexes. Proton NMR study suggests the preferential solvation of the four-coordinate species of the trans-α² complex to that of the α⁴ complex. It could be concluded that the stabilization of the base binding by the pickets is attributed to an intramolecular ligand–ligand interaction between the ligated base and the pickets rather than to the inhibition of the undesirable solvation on the active sites.     

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors

Changeux et al. (Changeux et al. C. R. Biol. 343:33–39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4β2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4β2 and αβγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.     

Camel lactoferrin, a transferrin-cum-lactoferrin: crystal structure of camel apolactoferrin at 2.6 A resolution and structural basis of its dual role

Camel lactoferrin is the first protein from the transferrin superfamily that has been found to display the characteristic functions of iron binding and release of lactoferrin as well as transferrin simultaneously. It was remarkable to observe a wide pH demarcation in the release of iron from two lobes. It loses 50 % iron at pH 6.5 and the remaining 50 % iron is released only at pH values between 4.0 and 2.0. Furthermore, proteolytically generated N and C-lobes of camel lactoferrin showed that the C-lobe lost iron at pH 6.5, while the N-lobe lost it only at pH less than 4.0. In order to establish the structural basis of this striking observation, the purified camel apolactoferrin was crystallized. The crystals belong to monoclinic space group C2 with unit cell dimensions a=175.8 A, b=80.9 A, c=56.4 A, beta=92.4 degrees and Z=4. The structure has been determined by the molecular replacement method and refined to an R-factor of 0.198 (R-free=0.268) using all the data in the resolution range of 20.0-2.6 A. The overall structure of camel apolactoferrin folds into two lobes which contain four distinct domains. Both lobes adopt open conformations indicating wide distances between the iron binding residues in the native iron-free form of lactoferrin. The dispositions of various residues of the iron binding pocket of the N-lobe of camel apolactoferrin are similar to those of the N-lobe in human apolactoferrin, while the corresponding residues in the C-lobe show a striking similarity with those in the C-lobes of duck and hen apo-ovotransferrins. These observations indicate that the N-lobe of camel apolactoferrin is structurally very similar to the N-lobe of human apolactoferrin and the structure of the C-lobe of camel apolactoferrin matches closely with those of the hen and duck apo-ovotransferrins. These observations suggest that the iron binding and releasing behaviour of the N-lobe of camel lactoferrin is similar to that of the N-lobe of human lactoferrin, whereas that of the C-lobe resembles those of the C-lobes of duck and hen apo-ovotransferrins. Hence, it correlates with the observation of the N-lobe of camel lactoferrin losing iron at a low pH (4.0-2.0) as in other lactoferrins. On the other hand, the C-lobe of camel lactoferrin loses iron at higher pH (7.0-6.0) like transferrins suggesting its functional similarity to that of transferrins. Thus, camel lactoferrin can be termed as half lactoferrin and half transferrin.     

Heterogeneity of rat hepatic Ah Receptor: Identification of two receptor forms which differ in their biochemical properties

In cytosol, the rat hepatic Ah receptor (AhR) appears to exist in two distinct forms (AhR^α, AhR^β) in similar concentration. The binding of ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) to AhR^α requires the receptor be in its oligomeric 8–10 to S conformation (bound to other protein subunits), while ligand binding to AhR^β can occur with the dissociated 5–6 S form. Occupancy of AhR^β by ligand (TCDD) protects it from salt-dependent inactivation; AhR^β is not inactivated by high salt conditions. The addition of molybdate to cytosol during tissue homogenization stabilized AhR^α against salt-dependent inactivation and subunit dissociation but did not prevent dissociation of AhR^β by high salt. Although the presence of molybdate appears to stabilize AhR^α in its oligomeric 8–10 S, it had no significant effect on the overall amount of TCDD:AhR complex which bound to its specific DNA recognition site, the dioxin responsive element (DRE). These results suggest that AhR^α, unlike AhR^β, is either unable to transform or bind to the DRE with high affinity.     

Assignments of human integrin α1I domain in the apo and Mg2+ bound states

The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg²⁺ bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination.     

Characterization of the extended carbohydrate binding site of concanavalin A: Specificity for interaction with the nonreducing termini of α-(1→2)-linked disaccharides

p-Nitrophenyl 2-O-α-d-galactopyranosyl-α-d-mannopyranoside and p-nitrophenyl 2-O-α-d-glucopyranosyl-α-d-mannopyranoside were synthesized and the interactions of these disaccharides with concanavalin A (con A) were characterized. The kinetics of binding of the galactopyranosyl-containing disaccharide to con A were found to be similar to those observed with monosaccharides in that monophasic time dependencies for binding were observed. The glucopyranosyl-containing disaccharide, however, exhibited biphasic time dependencies which were similar to those previously observed for the binding of p-nitrophenyl 2-O-α-d-mannopyranosyl-α-d-mannopyranoside to con A. These results support a model wherein the α-(1→2)-linked disaccharides which exhibit biphasic binding kinetics must be able to bind to con A in two different and mutually exclusive orientations. The ability to bind to con A in two orientations is shared by α-(1→2)-linked disaccharides in which both glycosyl residues can interact separately with the primary glycosyl binding site of con A. According to the model, the initial fast phase of the biphasic reaction reflects binding of the ligand in two orientations so that two complexes are formed in amounts determined by the relative values of the rate constants for formation of each complex. The subsequent slow phase is proposed to reflect a slow equilibration of the less stable complex to the thermodynamically more stable one. In the more stable complex, the glycosyl residue at the reducing end of the disaccharide occupies the primary glycosyl binding site. The added stability of this complex is attributed to extended interactions between con A and groups on the second glycosyl residue. An axial orientation of OH-2 of the second glycopyranosyl residue appears to be the most important determinant for the extended interaction.     

Molecular dynamics simulations of the photoactive protein nitrile hydratase

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, α and β. Hydrogen bonds between βArg-56 and αCys-114 sulfenic acid (αCEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength λ < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.