Active Site Hydrogen Bond Network Research Articles

Arginine-178 is an essential residue for ITPA function

The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the first pathological ITPA mutants identified is R178C (rs746930990), which causes a fatal infantile encephalopathy, termed developmental and epileptic encephalopathy 35 (DEE 35). The accumulation of noncanonical nucleotides such as inosine triphosphate (ITP), is suspected to affect RNA and/or interfere with normal nucleotide function, leading to development of DEE 35. Molecular dynamics simulations have shown that the very rare R178C mutation does not significantly perturb the overall structure of the protein, but results in a high level of structural flexibility and disrupts active-site hydrogen bond networks, while preliminary biochemical data indicate that ITP hydrolyzing activity is significantly reduced for the R178C mutant. Here we report Michaelis-Menten enzyme kinetics data for the R178C ITPA mutant and three other position 178 ITPA mutants. These data confirm that position 178 is essential for ITPA activity and even conservative mutation at this site (R178K) results in significantly reduced enzyme activity. Our data support that disruption of the active-site hydrogen bond network is a major cause of diminished ITP hydrolyzing activity for the R178C mutation. These results suggest an avenue for developing therapies to address DEE 35.

Substrate-Assisted Mechanism for the Degradation of N-Glycans by a Gut Bacterial Mannoside Phosphorylase

The unknown human gut bacterium mannoside phosphorylase (UhgbMP) is involved in the metabolization of eukaryotic N-glycans lining the intestinal epithelium, a factor associated with the onset and symptoms of inflammatory bowel diseases. In contrast with most glycoside phosphorylases, the putative catalytic acid of UhgbMP, Asp104, is far from the scissile glycosidic bond, challenging the classical Koshland mechanism. Using quantum mechanics/molecular mechanics metadynamics, we demonstrate that the enzyme operates by substrate-assisted catalysis via the 3-hydroxyl group of the mannosyl unit, following a 1S5/B2,5 → [B2,5]‡ → 0S2 conformational itinerary. Given the conservation of the active site hydrogen bond network across the family, this mechanism is expected to apply to other GH130 enzymes, as well as recently characterized mannoside phosphorylases with similar folds. Gaining insight into the catalytic reaction of these enzymes can aid the design of specific inhibitors to control interactions between gut microbes and the host.

A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site.

We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and we demonstrate its application in the high-throughput evolution of an oxygenase. A single round of selection followed by a facile growth assay enabled Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Structural modeling suggests precise reorganization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.

Molecular dynamics study of functionally relevant interdomain and active site interactions in the autotransporter esterase EstA from Pseudomonas aeruginosa

ABSTRACT Structural and dynamical properties of the autotransporter esterase EstA from bacterium Pseudomonas aeruginosa were studied using molecular dynamics (MD) simulations. Four different systems, including the full-length EstA enzyme inserted into a solvated 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayer, as well as the solvated isolated passenger domain of EstA were simulated without ligand and in complex with 4-hydroxyphenyloctanoate bound as tetrahedral intermediate. Detailed analysis of non-covalent interactions was performed based on 100 ns long MD simulations. It was found that active site interactions include not only the catalytic triad (Ser14, Asp286, His289), but also a three-residue oxyanion hole (backbone of Ser14, Gly92 and Asn147), hydrophobic residues involved in tetrahedral intermediate stabilisation, and residues participating in the active site hydrogen bond network. Moreover, interactions between protein domains were analysed and it was found that interacting residues are located on specific structures not usually found in GDSL hydrolases or autotransporter β barrels. In the case of full-length EstA enzyme, MD simulations point to specific interactions between the central and remote regions of the active site which are important for adequate intermediate stabilisation.

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network.

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔG Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

Structure of cytochrome P450 2B4 with an acetate ligand and an active site hydrogen bond network similar to oxyferrous P450cam

Superposition of the active site of acetate-bound P4502B4 and oxyferrous P450cam. Bond lengths between the heme iron, the sixth ligand, and the hydroxyl of the conserved threonine are shown. Note that different threonine rotamers form the hydrogen bonds to the acetate and oxygen. • Crystal structure of cytochrome P450 2B4 in complex with acetate • Two water molecules insert in the I-helix of the cytochrome P450 2B4 mutant active site • In cytochrome P450 2B4 acetate binds to the heme and side chain of Thr302. • The acetate bound active site of ferric cytochrome P450 2B4 resembles that of the oxygen bound P450 cam .

Dissecting the Side Chain Interaction Energies of the Active Site Hydrogen Bond Network in a Rhomboid Protease GlpG

The intramembrane rhomboid proteases are of particular interest due to their ability to hydrolyze a substrate peptide bond at the active site which is buried within the membrane. The high-resolution structure of the E. coli rhomboid protease GlpG revealed the catalytic dyad Ser201/His254, which is surrounded by a protein matrix and connected to bulk water via a narrow water channel. Although multiple crystal structures have been solved for GlpG, its catalytic mechanism is not clearly understood.

Enzymatic Activity and Thermodynamic Stability of Biliverdin IXβ Reductase Are Maintained by an Active Site Serine.

Biliverdin reductase IXβ (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.

A Molecular Dynamics and Quantum Mechanics/Molecular Mechanics Study of the Catalytic Reductase Mechanism of Methionine Sulfoxide Reductase A: Formation and Reduction of a Sulfenic Acid

The catalytic mechanism of MsrA in Mycobacterium tuberculosis, in which S-methionine sulfoxide (Met-O) is reduced to methionine (Met), has been investigated using docking, molecular dynamics (MD) simulations, and ONIOM (quantum mechanics/molecular mechanics) methods. In addition, the roles of specific active site residues, including an aspartyl (Asp87) near the recycling cysteine, tyrosyls (Tyr44 and Tyr92), and glutamyl (Glu52), have been examined, as well as the general effects of the protein and active site on the nature and properties of mechanistic intermediates. The mechanism is initiated by the transfer of a proton from the catalytic cysteine's thiol (Cys13SH) via a bridging water to the R group carboxylate of Glu52. The now anionic sulfur of Cys13 nucleophilically attacks the substrate's sulfur with concomitant transfer of a proton from Glu52 to the sulfoxide oxygen, generating a sulfurane. The active site enhances the proton affinity of the sulfurane oxygen, which can readily accept a proton from the phenolic hydroxyls of Tyr44 or Tyr92 to give a sulfonium cation. Subsequently, Asp87 and the recycling cysteine (Cys154) can facilitate nucleophilic attack of a solvent water at the Cys13S center of the sulfonium to give a sulfenic acid (Cys13SOH) and Met. For the subsequent reduction of Cys13SOH with intramolecular disulfide bond formation, Asp87 can help facilitate nucleophilic attack of Cys154S at the sulfur of Cys13SOH by deprotonating its thiol. This reduction is found likely to occur readily upon suitable positioning of the active site hydrogen bond network and the sulfur centers of both Cys13 and Cys154. The calculated rate-limiting barrier is in good agreement with experiment.

Water in the Active Site of Ketosteroid Isomerase

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.

Identification by nuclear magnetic resonance spectroscopy of an active-site hydrogen-bond network in human monoacylglycerol lipase (hMGL): implications for hMGL dynamics, pharmacological inhibition, and catalytic mechanism.

Intramolecular hydrogen bonding is an important determinant of enzyme structure, catalysis, and inhibitor action. Monoacylglycerol lipase (MGL) modulates cannabinergic signaling as the main enzyme responsible for deactivating 2-arachidonoylglycerol (2-AG), a primary endocannabinoid lipid messenger. By enhancing tissue-protective 2-AG tone, targeted MGL inhibitors hold therapeutic promise for managing pain and treating inflammatory and neurodegenerative diseases. We report study of purified, solubilized human MGL (hMGL) to explore the details of hMGL catalysis by using two known covalent hMGL inhibitors, the carbamoyl tetrazole AM6701 and N-arachidonoylmaleimide (NAM), that act through distinct mechanisms. Using proton nuclear magnetic resonance spectroscopy (NMR) with purified wild-type and mutant hMGLs, we have directly observed a strong hydrogen-bond network involving Asp239 and His269 of the catalytic triad and neighboring Leu241 and Cys242 residues. hMGL inhibition by AM6701 alters this hydrogen-bonding pattern through subtle active-site structural rearrangements without influencing hydrogen-bond occupancies. Rapid carbamoylation of hMGL Ser122 by AM6701 and elimination of the leaving group is followed by a slow hydrolysis of the carbamate group, ultimately regenerating catalytically competent hMGL. In contrast, hMGL titration with NAM, which leads to cysteine alkylation, stoichiometrically decreases the population of the active-site hydrogen bonds. NAM prevents reformation of this network, and in this manner inhibits hMGL irreversibly. These data provide detailed molecular insight into the distinctive mechanisms of two covalent hMGL inhibitors and implicate a hydrogen-bond network as a structural feature of hMGL catalytic function.

Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site

Hydrogen bond networks are key elements of biological structure and function. Nevertheless, their structural properties are challenging to assess within complex macromolecules. Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures, and static crystallographic models provide limited information regarding the dynamical coupling within hydrogen bond networks. We have brought together 1.1-1.3 A resolution X-ray crystallography, (1)H NMR, site-directed mutagenesis, and deuterium isotope effects on the geometry and chemical shifts of hydrogen-bonded protons to probe the conformational coupling of hydrogen bonds donated by Y16 and D103 in the oxyanion hole of bacterial ketosteroid isomerase. Our results suggest a robust physical coupling of the equilibrium structures of these two hydrogen bonds such that a lengthening of one hydrogen bond by as little as 0.01 A results in a shortening of the neighbor by a similar magnitude. Furthermore, the structural rearrangements detected by NMR in response to mutations within the active site hydrogen bond network can be explained on the basis of the observed coupling. The results herein elucidate fundamental structural properties of hydrogen bonds within the idiosyncratic environment of an enzyme active site and provide a foundation for future experimental and computational explorations of the role of coupled motions within hydrogen bond networks.

Saturation mutagenesis of Asn152 reveals a substrate selectivity switch in P99 cephalosporinase

In class C beta-lactamases, the strictly conserved Asn152 forms part of an extended active-site hydrogen-bonding network. To probe its role in catalysis, all 19 mutants of Enterobacter cloacae P99 cephalosporinase Asn152 were simultaneously constructed and screened in Escherichia coli for their in vivo activity. The screen identified the previously uncharacterized mutants Asn152Ser, Asn152Thr, and Asn152Gly, which possess significant activity and altered substrate selectivity. In vitro measurement of Michaelis-Menten kinetic constants revealed that the Asn152Ser mutation causes a selectivity switch for penicillin G versus cefoxitin. Asn152Thr showed a 63-fold increase in k (cat) for oxacillin, a slow substrate for wild-type cephalosporinase. The results contribute to a growing body of data showing that mutation of highly conserved residues in the active site can result in substrate selectivity changes. The library screening method presented here would be applicable to substrate selectivity determination in other readily screenable enzymes.

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

Crystal Structure of Pseudomonas aeruginosa SPM-1 Provides Insights into Variable Zinc Affinity of Metallo-β-lactamases

Metallo-beta-lactamases (mbetals) confer broad-spectrum resistance to beta-lactam antibiotics upon host bacteria and escape the action of existing beta-lactamase inhibitors. SPM-1 is a recently discovered mbetal that is distinguished from related enzymes by possession of a substantial central insertion and by sequence variation at positions that maintain active site structure. Biochemical data show SPM-1 to contain two Zn2+ sites of differing affinities, a phenomenon that is well documented amongst mbetals but for which a structural explanation has proved elusive. Here, we report the crystal structure of SPM-1 to 1.9 A resolution. The structure reveals SPM-1 to lack a mobile loop implicated in substrate binding by related mbetals and to accommodate the central insertion in an extended helical interdomain region. Deleting this had marginal effect upon binding and hydrolysis of a range of beta-lactams. These data suggest that the interactions of SPM-1 with substrates differ from those employed by other mbetals. SPM-1 as crystallised contains a single Zn2+. Both the active site hydrogen-bonding network and main-chain geometry at Asp120, a key component of the binding site for the second zinc ion, differ significantly from previous mbetal structures. We propose that variable interactions made by the Asp120 carbonyl group modulate affinity for a second Zn2+ equivalent in mbetals of the B1 subfamily. We further predict that SPM-1 possesses the capacity to evolve variants of enhanced catalytic activity by point mutations altering geometry and hydrogen bonding in the vicinity of the second Zn2+ site.

Crystal structures of the G139A, G139A?NO and G143H mutants of human heme oxygenase-1. A finely tuned hydrogen-bonding network controls oxygenase versus peroxidase activity

Conserved glycines, Gly139 and Gly143, in the distal helix of human heme oxygenase-1 (HO-1) provide the flexibility required for the opening and closing of the heme active site for substrate binding and product dissociation during HO-1 catalysis. Earlier mutagenesis work on human HO-1 showed that replacement of either Gly139 or Gly143 suppresses heme oxygenase activity and, in the case of the Gly139 mutants, increases peroxidase activity (Liu et al. in J. Biol. Chem. 275:34501, 2000). To further investigate the role of the conserved distal helix glycines, we have determined the crystal structures of the human HO-1 G139A mutant, the G139A mutant in a complex with NO, and the G143H mutant at 1.88, 2.18 and 2.08 A, respectively. The results confirm that fine tuning of the previously noted active-site hydrogen-bonding network is critical in determining whether heme oxygenase or peroxidase activity is observed.

Identification of residues critical for catalysis in a class C beta-lactamase by combinatorial scanning mutagenesis.

Despite their clinical importance, the mechanism of action of the class C beta-lactamases is poorly understood. In contrast to the class A and class D beta-lactamases, which contain a glutamate residue and a carbamylated lysine in their respective active sites that are thought to serve as general base catalysts for beta-lactam hydrolysis, the mechanism of activation of the serine and water nucleophiles in the class C enzymes is unclear. To probe for residues involved in catalysis, the class C beta-lactamase from Enterobacter cloacae P99 was studied by combinatorial scanning mutagenesis at 122 positions in and around the active site. Over 1000 P99 variants were screened for activity in a high-throughput in vivo antibiotic resistance assay and sequenced by 96-capillary electrophoresis to identify residues that are important for catalysis. P99 mutants showing reduced capability to convey antibiotic resistance were purified and characterized in vitro. The screen identified an active-site hydrogen-bonding network that is key to catalysis. A second cluster of residues was identified that likely plays a structural role in the enzyme. Otherwise, residues not directly contacting the substrate showed tolerance to substitution. The study lends support to the notion that the class C beta-lactamases do not have a single residue that acts as the catalytic general base. Rather, catalysis is affected by a hydrogen-bonding network in the active site, suggesting a possible charge relay system.

Ab initio QM/MM dynamics simulation of the tetrahedral intermediate of serine proteases: insights into the active site hydrogen-bonding network.

Ab initio QM/MM dynamics simulation is employed to examine the stability of the tetrahedral intermediate during the deacylation step in elastase-catalyzed hydrolysis of a simple peptide. An extended quantum region includes the catalytic triad, the tetrahedral structure, and the oxyanion hole. The calculations indicate that the tetrahedral intermediate of serine proteases is a stable species on the picosecond time scale. On the basis of geometrical and dynamical properties, and in agreement with many experimental and theoretical studies, it is suggested that the crucial hydrogen bonds involved in stabilizing this intermediate are between Asp-102 and His-57 and between the charged oxygen of the intermediate and the backbone N-H group of Gly-193 in the oxyanion hole. The mobility of the imidazolium ring between O(w) and O(gamma), two of the oxygens of the tetrahedral structure, shows how the intermediate could proceed toward the product state without a "ring-flip mechanism", proposed earlier on the basis of NMR data. In addition to the proposed C(epsilon)(1)-H.O hydrogen bond between the imidazolium ring and the backbone carbonyl of Ser-214, we observe an alternative C(epsilon)(1)-H.O hydrogen bond with the backbone carbonyl of Thr-213, that can stabilize the intermediate during the imidazolium movement. Proton hopping occurs between Asp-102 and His-57 during the simulation. The proton is, however, largely localized on the nitrogen, and hence it does not participate in a low-barrier hydrogen bond. The study also suggests factors that may be implicated in product release: breaking the hydrogen bond of the charged oxygen with the backbone of Ser-195 in the oxyanion hole and a loop opening between residues 216-225 that enables the breaking of a hydrogen bond in subsite S(3).

Influence of the Distal His in Imparting Imidazolate Character to the Proximal His in Heme Peroxidase: 1H NMR Spectroscopic Study of Cyanide-Inhibited His42→Ala Horseradish Peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn 2+, Fe 3+ or Fe 2+ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn 2+, Fe 3+ and 1H in the presence of Fe 2+ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe 2+SOD and Fe 2+(Mn)SOD by 15N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe 2+ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH − coordinated to the metal ion.