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1.
Methods Enzymol ; 577: 119-58, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27498637

RESUMO

In this chapter, I discuss combined quantum mechanics (QM) and molecular mechanics (MM; QM/MM) calculations for proteins. In QM/MM, a small but interesting part of the protein is treated by accurate QM methods, whereas the remainder is treated by faster MM methods. The prime problems with QM/MM calculations are bonds between the QM and MM systems, the selection of the QM system, and the local-minima problem. The two first problems can be solved by the big-QM approach, including in the QM calculation all groups within 4.5-6Å of the active site and all buried charges in the protein. The third problem can be solved by calculating free energies. It is important to study QM/MM energy components to ensure that the results are stable and reliable. They can also be used to understand the reaction and the effect of the surroundings, eg, by dividing the catalytic effect into bonded, van der Waals, electrostatic, and geometric components and to deduce which parts of the protein contribute most to the catalysis. It should be ensured that the QM calculations are reliable and converged by extending the basis set to quadruple-zeta quality, including a proper treatment of dispersion, as well as years experience and method development calculations with both pure and hybrid density functional theory methods. If the latter give differing results, calibration with high-level QM methods is needed. Reactions that change the net charge should be avoided. QM/MM calculations can be combined with experimental methods.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Teoria Quântica , Termodinâmica , Animais , Humanos
2.
J Mol Biol ; 261(4): 586-96, 1996 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-8794878

RESUMO

The geometry of several realistic models of the metal coordination sphere in the blue copper proteins has been optimised using high-level quantum chemical methods. The results show that the optimal vacuum structure of the Cu(II) models is virtually identical to the crystal structure of oxidised blue copper proteins. For the reduced forms, the optimised structure seems to be more tetrahedral than the one found in the proteins, but the energy difference between the two geometries is less than 5 kJ/mol, i.e. within the error limits of the method. Thus, the results raise strong doubts against hypotheses (entatic state and the induced-rack theory) suggesting that blue copper proteins force the oxidised metal coordination sphere into a structure similar to that preferred by Cu(I) in order to minimise the reorganisation energy of the electron transfer reaction. Instead, a small reorganisation energy seems to be reached by an appropriate choice of metal ligands. In particular, the cysteine thiolate ligand appears to be crucial, changing the preferred geometry of the oxidised complexes from square-planar to a more trigonal geometry.


Assuntos
Proteínas de Bactérias/química , Cobre/química , Modelos Moleculares , Ligantes , Oxirredução , Conformação Proteica , Teoria Quântica , Vácuo
3.
Biophys J ; 77(5): 2777-87, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10545376

RESUMO

The relative energies of different coordination modes (bidentate, monodentate, syn, and anti) of a carboxylate group bound to a zinc ion have been studied by the density functional method B3LYP with large basis sets on realistic models of the active site of several zinc proteins. In positively charged four-coordinate complexes, the mono- and bidentate coordination modes have almost the same energy (within 10 kJ/mol). However, if there are negatively charged ligands other than the carboxylate group, the monodentate binding mode is favored. In general, the energy difference between monodentate and bidentate coordination is small, 4-24 kJ/mol, and it is determined more by hydrogen-bond interactions with other ligands or second-sphere groups than by the zinc-carboxylate interaction. Similarly, the activation energy for the conversion between the two coordination modes is small, approximately 6 kJ/mol, indicating a very flat Zn-O potential surface. The energy difference between syn and anti binding modes of the monodentate carboxylate group is larger, 70-100 kJ/mol, but this figure again strongly depends on interactions with second-sphere molecules. Our results also indicate that the pK(a) of the zinc-bound water ligand in carboxypeptidase and thermolysin is 8-9.


Assuntos
Ácidos Carboxílicos/metabolismo , Proteínas/química , Proteínas/metabolismo , Teoria Quântica , Zinco , Ácidos Carboxílicos/química , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Hidróxidos/metabolismo , Ligantes , Ligação Proteica , Prótons , Estereoisomerismo , Termolisina/química , Termolisina/metabolismo , Água/metabolismo
4.
Protein Sci ; 4(6): 1124-32, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7549877

RESUMO

Theoretical computations (molecular dynamics and combined quantum chemical and molecular mechanical geometry optimizations) have been performed on horse liver alcohol dehydrogenase. The results provide evidence that Glu-68, a highly conserved residue located 0.47 nm from the catalytic zinc ion, may intermittently coordinate to the zinc ion. Structures with Glu-68 coordinated to the zinc ion are almost as stable as structures with Glu-68 at the crystal position and the barrier between the two configurations of Glu-68 is so low that it can readily be bypassed at room temperature. There is a cavity behind the zinc ion that seems to be tailored to allow such coordination of Glu-68 to the zinc ion. It is suggested that Glu-68 may facilitate the exchange of ligands in the substrate site by coordinating to the zinc ion when the old ligand dissociates.


Assuntos
Álcool Desidrogenase/química , Ácido Glutâmico/química , Zinco/química , Álcool Desidrogenase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Simulação por Computador , Sequência Conservada , Cavalos , Fígado/enzimologia , Modelos Moleculares
5.
Protein Sci ; 7(12): 2659-68, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9865961

RESUMO

The inner-sphere reorganization energy for several copper complexes related to the active site in blue-copper protein has been calculated with the density functional B3LYP method. The best model of the blue-copper proteins, Cu(Im)2(SCH3)(S(CH3)2)(0/+), has a self-exchange inner-sphere reorganization energy of 62 kJ/mol, which is at least 120 kJ/mol lower than for Cu(H2O)4(+/2+). This lowering of the reorganization energy is caused by the soft ligands in the blue-copper site, especially the cysteine thiolate and the methionine thioether groups. Soft ligands both make the potential surfaces of the complexes flatter and give rise to oxidized structures that are quite close to a tetrahedron (rather than tetragonal). Approximately half of the reorganization energy originates from changes in the copper-ligand bond lengths and half of this contribution comes from the Cu-S(Cys) bond. A tetragonal site, which is present in the rhombic type 1 blue-copper proteins, has a slightly higher (16 kJ/mol) inner-sphere reorganization energy than a trigonal site, present in the axial type 1 copper proteins. A site with the methionine ligand replaced by an amide group, as in stellacyanin, has an even higher reorganization energy, about 90 kJ/mol.


Assuntos
Proteínas de Bactérias/química , Cobre/metabolismo , Modelos Químicos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cobre/química , Modelos Moleculares
6.
J Comput Chem ; 28(6): 1083-90, 2007 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-17279548

RESUMO

Localized multipole moments up to the fifth moment as well as localized dipole polarizabilities are calculated with the MpProp and the newly developed LoProp methods for a total of 20 molecules, predominantly derived from amino acids. A comparison of electrostatic potentials calculated from the multipole expansion obtained by the two methods with ab initio results shows that both methods reproduce the electrostatic interaction with an elementary charge with a mean absolute error of approximately 1.5 kJ/mol at contact distance and less than 0.1 kJ/mol at distances 2 A further out when terms up to the octupole moments are included. The polarizabilities are tested with homogenous electric fields and are found to have similar accuracy. The MpProp method gives better multipole moments unless diffuse basis sets are used, whereas LoProp gives better polarizabilities.

7.
Proteins ; 21(1): 40-56, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7716168

RESUMO

A detailed parameterization is presented of a zinc ion with one histidine and two cysteinate ligands, together with one or two water, hydroxide, aldehyde, alcohol, or alkoxide ligands. The parameterization is tailored for the active site of alcohol dehydrogenase and is obtained entirely from quantum chemical computations. The force-field reproduces excellently the geometry of quantum chemically optimized zinc complexes as well as the crystallographic geometry of the active site of alcohol dehydrogenase and small organic structures. The parameterization is used in molecular dynamics simulations and molecular mechanical energy minimizations of alcohol dehydrogenase with a four- or five-coordinate catalytic zinc ion. The active-site zinc ion seems to prefer four-coordination over five-coordination by at least 36 kJ/mol. The only stable binding site of a fifth ligand at the active-site zinc ion is opposite to the normal substrate site, in a narrow cavity behind the zinc ion. Only molecules of the size of water or smaller may occupy this site. There are large fluctuations in the geometry of the zinc coordination sphere. A four-coordinate water molecule alternates frequently (every 7 ps) between the substrate site and the fifth binding site and even two five-coordinate water molecules may interchange ligation sites without prior dissociation. Ligand exchange at the zinc ion probably proceeds by a dissociative mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Álcool Desidrogenase/química , Zinco/química , Íons , Ligantes , Matemática , Estrutura Molecular , Fenômenos Físicos , Física
8.
J Comput Aided Mol Des ; 10(2): 153-64, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8741019

RESUMO

The coordination number of the catalytic zinc ion in alcohol dehydrogenase has been studied by integrated ab initio quantum-chemical and molecular mechanics geometry optimisations involving the whole enzyme. A four-coordinate active-site zinc ion is 100-200 kJ/mol more stable than a five-coordinate one, depending on the ligands. The only stable binding site for a fifth ligand at the zinc ion is opposite to the normal substrate site, in a small cavity buried behind the zinc ion. The zinc coordination sphere has to be strongly distorted to accommodate a ligand in this site, and the ligand makes awkward contacts with surrounding atoms. Thus, the results do not support proposals attributing an important role to five-coordinate zinc complexes in the catalytic mechanism of alcohol dehydrogenase. The present approach makes it possible also to quantify the strain induced by the enzyme onto the zinc ion and its ligands; it amounts to 42-87 kJ/mol for four-coordinate active-site zinc ion complexes and 131-172 kJ/mol for five-coordinate ones. The four-coordinate structure with a water molecule bound to the zinc ion is about 20 kJ/mol less strained than the corresponding structure with a hydroxide ion, indicating that the enzyme does not speed up the reaction by forcing the zinc coordination sphere into a structure similar to the reaction intermediates.


Assuntos
Álcool Desidrogenase/química , Conformação Proteica , Zinco/análise , Álcool Desidrogenase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria , Ligantes , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Teoria Quântica , Software , Zinco/química
9.
J Biol Inorg Chem ; 4(1): 99-110, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10499107

RESUMO

Quantum chemical geometry optimizations have been performed on realistic models of the active site of myoglobin using density functional methods. The energy of the hydrogen bond between the distal histidine residue and CO or O2 has been estimated to be 8 kJ/mol and 32 kJ/mol, respectively. This 24 kJ/mol energy difference accounts for most of the discrimination between CO and O2 by myoglobin (about 17 kJ/mol). Thus, steric effects seem to be of minor importance for this discrimination. The Fe-C and C-O vibrational frequencies of CO-myoglobin have also been studied and the results indicate that CO forms hydrogen bonds to either the distal histidine residue or a water molecule during normal conditions. We have made several attempts to optimise structures with the deprotonated nitrogen atom of histidine directed towards CO. However, all such structures lead to unfavourable interactions between the histidine and CO, and to vCO frequencies higher than those observed experimentally.


Assuntos
Monóxido de Carbono/metabolismo , Ligação de Hidrogênio , Mioglobina/química , Mioglobina/metabolismo , Oxigênio/metabolismo , Simulação por Computador , Histidina/química , Histidina/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Porfirinas/química , Porfirinas/metabolismo , Vibração
10.
J Am Chem Soc ; 123(32): 7866-76, 2001 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-11493060

RESUMO

The dimeric Cu(A) site found in cytochrome c oxidase and nitrous oxide reductase has been studied with the density functional B3LYP method. We have optimized the structure of the realistic (Im)(S(CH(3))(2))Cu(SCH(3))(2)Cu(Im)(CH(3)CONHCH(3)) model in the fully reduced, mixed-valence, and fully oxidized states. The optimized structures are very similar to crystal structures of the protein, which shows that the protein does not strain the site significantly. Instead, inorganic model complexes of the protein site are strained by the macrocyclic connections between the ligand models. For the mixed-valence (Cu(I)+Cu(II)) state, two distinct equilibrium structures were found, one with a short Cu-Cu distance, 248 pm, similar to the protein structure, and one with a longer distance, 310 pm, similar to what is found in inorganic models. In the first state, the unpaired electron is delocalized over both copper ions, whereas in the latter, it is more localized to one of the ions. The two states are nearly degenerate. The potential energy surfaces for the Cu-Cu, Cu-S(Met), and Cu-O interactions are extremely flat. In fact, all three distances can be varied between 230 and 310 pm at an expense in energy of less than 8 kJ/mol, which explains the large variation observed in crystal structures for these interactions. Inclusion of solvation effects does not change this significantly. Therefore, we can conclude that a variation in these distances can change the reduction potential of the Cu(A) site by at most 100 mV. The model complex has a reorganization energy of 43 kJ/mol, 20 kJ/mol lower than for a monomeric blue-copper site. This lowering is caused by the delocalization of the unpaired electron in the mixed-valence state.

11.
Proteins ; 36(2): 157-74, 1999 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10398364

RESUMO

Free energy perturbations have been performed on two blue copper proteins, plastocyanin and nitrite reductase. By changing the copper coordination geometry, force constants, and charges, we have estimated the maximum energy with which the proteins may distort the copper coordination sphere. By comparing this energy with the quantum chemical energy cost for the same perturbation on the isolated copper complex, various hypotheses about protein strain have been tested. The calculations show that the protein can only modify the copper-methionine bond length by a modest amount of energy-<5 kJ/mol-and they lend no support to the suggestion that the quite appreciable difference in the copper coordination geometry encountered in the two proteins is a result of the proteins enforcing different Cu-methionine bond lengths. On the contrary, this bond is very flexible, and neither the geometry nor the electronic structure change appreciably when the bond length is changed. Moreover, the proteins are rather indifferent to the length of this bond. Instead, the Cu(II) coordination geometries in the two proteins represent two distinct minima on the potential surface of the copper ligand sphere, characterized by different electronic structures, a tetragonal, mainly sigma-bonded, structure in nitrite reductase and a trigonal, pi-bonded, structure in plastocyanin. In vacuum, the structures have almost the same energy, and they are stabilized in the proteins by a combination of geometric and electrostatic interactions. Plastocyanin favors the bond lengths and electrostatics of the trigonal structure, whereas in nitrite reductase, the angles are the main discriminating factor. Proteins 1999;36:157-174.


Assuntos
Proteínas de Bactérias/química , Cobre/metabolismo , Bactérias , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Calibragem , Cátions/química , Cátions/metabolismo , Simulação por Computador , Cobre/química , Ligantes , Modelos Moleculares , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredução , Plastocianina/química , Plastocianina/metabolismo , Conformação Proteica , Eletricidade Estática , Termodinâmica , Água/química , Água/metabolismo
12.
Protein Eng ; 12(10): 851-6, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10556245

RESUMO

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.


Assuntos
Coenzimas/metabolismo , Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Motivos de Aminoácidos/genética , Sítios de Ligação/genética , Clonagem Molecular , Simulação por Computador , Meios de Cultura , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Geobacillus stearothermophilus/genética , L-Lactato Desidrogenase/metabolismo , Metanol/farmacologia , Modelos Moleculares , NAD/metabolismo , NADP/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
13.
J Biol Inorg Chem ; 4(5): 654-63, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10550695

RESUMO

The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S(Met) bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site.


Assuntos
Proteínas de Bactérias/química , Metionina/química , Ligantes , Oxirredução , Teoria Quântica
14.
Inorg Chem ; 40(11): 2509-19, 2001 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-11350228

RESUMO

Models of several types of iron-sulfur clusters (e.g., Fe(4)S(4)(SCH(3))(4)(2-/3-/4-)) have been studied with the density functional B3LYP method and medium-sized basis sets. In a vacuum, the inner-sphere reorganization energies are 40, 76, 40, 62, 43, and 42 kJ/mol for the rubredoxin, [2Fe-2S] ferredoxin, Rieske, [4Fe-4S] ferredoxin, high-potential iron protein, and desulfoferrodoxin models, respectively. The first two types of clusters were also studied in the protein, where the reorganization energy was approximately halved. This change is caused by the numerous NH.S(Cys) hydrogen bonds to the negatively charged iron-sulfur cluster, giving rise to a polar local environment. The reorganization energy of the iron-sulfur clusters is low because the iron ions retain the same geometry and coordination number in both oxidation states. Cysteine ligands give approximately the same reorganization energy as imidazole, but they have the advantage of stabilizing a lower coordination number and giving more covalent bonds and therefore more effective electron-transfer paths.


Assuntos
Ferredoxinas/química , Ferro/química , Enxofre/química , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Termodinâmica
15.
J Biol Inorg Chem ; 5(5): 565-74, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11085647

RESUMO

Theoretical investigations of the structure and function of the blue copper proteins are described. We have studied the optimum vacuum geometry of oxidised and reduced copper sites, the relative stability of trigonal and tetragonal Cu(II) structures, the relation between the structure and electronic spectra, the reorganisation energy, and reduction potentials. Our calculations give no support to the suggestion that strain plays a significant role in the function of these proteins; on the contrary, our results show that the structures encountered in the proteins are close to their optimal vacuum geometries (within 7 kJ/mol). We stress the importance of defining what is meant by strain and of quantifying strain energies or forces in order to make strain hypotheses testable.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Cobre/metabolismo , Transporte de Elétrons , Elétrons , Modelos Moleculares , Oxirredução , Conformação Proteica , Teoria Quântica , Relação Estrutura-Atividade
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