Effect of local environment and protein on the mechanism of action of superoxide dismutase

Quantum mechanical simulations of the mechanism of action of superoxide dismutase (SOD) indicate that the presence of Arg-141 in the active site of the enzyme is responsible for the formation of an intermediate complex between superoxide and the enzyme in which the copper is not reduced. The analysis of the local environmental effects of Arg-141 shows that this residue prevents the reduction of copper by forming a hydrogen bond to superoxide and by generating an electric field in the active site that opposes the transfer of an electron from superoxide to copper. The protein enhances the effect of the opposing field generated by Arg-141. Local changes in the environment of the copper ion, simulated by stretching the Cu-NE2 (His-61) bond also do not induce an electron transfer from superoxide to copper. The protein increases the energies required for this stretch through the electric field it generates near the active site. These results are further support for the new proposed mechanism of action of SOD which is based on the inability of superoxide to reduce the cupric ion in the enzyme.     

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O₂ and H₂O₂. SOD also reacts with H₂O₂, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO₂) and H₂O₂, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.     

Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Toward the engineering of a super efficient enzyme

The catalytic activity of a mutant of Photobacterium leiognathi Cu, Zn superoxide dismutase in which the Glu59 residue, conserved in most bacterial variants of the enzyme, has been replaced by glutamine was investigated by pulse radiolysis. At neutral pH the enzyme was found to have a kcat/KM of 1.0 +/- 0.1 x 10(10) M-1s-1 the highest value ever found for any superoxide dismutase. Brownian dynamics simulation suggests that such a high value is due to an enhanced substrate attraction by the modified electric field distribution. The mutant is also characterized by an active-site widely accessible for the solvent, since iodide is able to interact with the copper atom with an affinity constant twice as high as that found in the native enzyme. The large solvent accessible surface of the copper site together with a favorable distribution of the protein-generated electric field gives rise to the most efficient enzyme ever found with activity close to the diffusion limit.     

Structure and dynamics of the active site gorge of acetylcholinesterase: synergistic use of molecular dynamics simulation and X-ray crystallography.

The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.     

Dioxygen,an unexpected carbonic anhydrase ligand

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, grouped into seven different classes, which catalyze the reaction of CO₂ hydration to bicarbonate and protons. All of the fifteen human isoforms reported to date belong to the α-class and contain zinc as a cofactor. The structure of human Zn,Cu-CA II has been solved which contains a copper ion bound at its N-terminal, coordinated to His4 and His64. In the active site a dioxygen molecule is coordinated to the zinc ion. Since dioxygen is a rather unexpected CA ligand, molecular dynamics (MD) simulations were performed which suggested a superoxide character of the zinc bound O₂.     

变性过程中铜锌超氧化物歧化酶的活性通道

在酶的盐酸胍变性和热变性过程中,尝试采用电荷传递反应分析方法和电子自旋共振方法考察了酶活性部位的构象变化。酶活力与构象的变化行为表明,酶的活性部位通道先于酶分子的整体构象而发生变化,它是与酶的失活同时发生的。尽管酶活性部位中的金属离子保证了酶较高的稳定性,但酶的活性部位,特别是活性通道仍然是相对脆弱的。     

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.     

The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H₂ has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H₂ with a limiting rate constant of 5.3 s⁻¹ and a dissociation constant K_d of 525 μm; both k_red and k_red/K_d, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H₂, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site ^63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H₂ oxidation is proposed which involves initial binding of H₂ to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.     

Energetics of the proposed rate-determining step of the glyoxalase I reaction.

The proposed rate-limiting step of the reaction catalyzed by glyoxalase I is the proton abstraction from the C1 carbon atom of the substrate by a glutamate residue, resulting in a high-energy enolate intermediate. This proton transfer reaction was modelled using molecular dynamics and free energy perturbation simulations, with the empirical valence bond method describing the potential energy surface of the system. The calculated rate constant for the reaction is approximately 300-1500 s(-1) with Zn2+, Mg2+ or Ca2+ bound to the active site, which agrees well with observed kinetics of the enzyme. Furthermore, the results imply that the origin of the catalytic rate enhancement is mainly associated with enolate stabilization by the metal ion.     

Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases.

The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.     

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase (ACMSD): A structural and mechanistic unveiling

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu‐substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super‐hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue‐bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand‐bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2015; 83:178–187. © 2014 Wiley Periodicals, Inc.     

The mechanism of superoxide dismutase functioning: a multicenter model

A new model of superoxide dismutase (SOD) functioning has been proposed on the basis of recent data. This model takes into account both experimental data and results of quantum mechanics calculations. One substrate molecule (superoxide radical) binds copper ion in active center of SOD, and the second superoxide radical interacts with a peripheral region of the enzyme. Uncoupled electron from the active center is transferred to the peripheral superoxide anion. This results in formation of oxygen molecule in the active center, and of hydrogen peroxide molecule in the peripheral region.     

Chemical modification of the CuA center in cytochrome c oxidase by sodium p-(hydroxymercuri)benzoate

Cytochrome c oxidase contains a copper ion electron-transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-(hydroxymercuri) benzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center that has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure,functionality and tuning up of laccases for lignocellulose and other industrial applications

Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and involves a series of electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be expanded by means of supplementary mediators that essentially function as vehicles for electron transfer. Comparisons of amino acid sequences and structural traits of selected laccases reveal conservation of the active site trinuclear center geometry but differences in loop conformations. We also evaluate the features and regions of laccases in relation to modification and evolution of laccases for various industrial applications including lignocellulosic biomass processing.     

Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.     

Probing the Structural Basis for Enzyme-Substrate Recognition In Cu,Zn Superoxide Dismutase

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu. Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stcreochemistry. supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu132, Glu133, Lys136, Thr137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.     

Redox-coupled proton pumping in cytochrome c oxidase: further insights from computer simulation

The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a3 (PRDa3) for the first time. The results from these molecular dynamics simulations indicate that the PRDa3 can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa3 side chain isomerization and the proton translocation between Glu242 and the PRDa3 site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa3 site.     

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.