Biomedical subjects

Ulf Ryde

Publications and source records attributed to Ulf Ryde.

35 records · Page 2Linked to original sources

PubMed2005

A comparative reactivity study of microperoxidases based on hemin, mesohemin and deuterohemin.

Three microperoxidases--hemin-6(7)-gly-gly-his methyl ester (HGGH), mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH)--have been prepared as models for heme-containing peroxidases by condensation of glycyl-glycyl-L-histidine methyl ester with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. The three microperoxidases differ in two substituents, R, of the protoporphyrin IX framework (HGGH: R=vinyl, MGGH: R=ethyl, DGGH: R=H). X-band and high field EPR spectra show that the microperoxidases exhibit spectroscopic properties similar to those of metmyoglobin, i.e. a high spin ferric S=5/2 signal at g(perpendicular)=6 and g parallel)=2 and an estimated D value of 7.5+/-1cm(-1). The catalytic activities of the microperoxidases towards K4[Fe(CN)6], L-tyrosine methyl ester and 2,2'-azino(bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) have been investigated. It was found that all three microperoxidases exhibit peroxidase activity and that the reactions follow the generally accepted peroxidase reaction scheme [Biochem. J. 145 (1975) 93-103] with the exception that the initial formation of a Compound I analogue is the rate-limiting step for the whole process. The general activity trend was found to be MGGH approximately DGGH>HGGH. For each microperoxidase, DFT calculations (B3LYP) were made on the reactions of compounds 0, I and II with H+, e- and H+ + e-, respectively, in order to probe the possible relationship between the nature of the 2- and 4-substituents of the hemin and the observed reactivity. The computational modeling indicates that the relative energy differences are very small; solvation and electrostatic effects may be factors that decide the relative activities of the microperoxidases.

Benzothiazoles↗

PubMed2004

The protonation status of compound II in myoglobin, studied by a combination of experimental data and quantum chemical calculations: quantum refinement.

Treatment of met-myoglobin (FeIII) with H2O2 gives rise to ferryl myoglobin, which is closely related to compound II in peroxidases. Experimental studies have given conflicting results for this species. In particular, crystallographic and extended x-ray absorption fine-structure data have shown either a short (approximately 170 pm) or a longer (approximately 190 pm) Fe-O bond, indicating either a double or a single bond. We here present a combined experimental and theoretical investigation of this species. In particular, we use quantum refinement to re-refine a crystal structure with a long bond, using 12 possible states of the active site. The states differ in the formal oxidation state of the iron ion and in the protonation of the oxygen ligand (O2-, OH-, or H2O) and the distal histidine residue (with a proton on Ndelta1, Nepsilon2, or on both atoms). Quantum refinement is essentially standard crystallographic refinement, where the molecular-mechanics potential, normally used to supplement the experimental data, is replaced by a quantum chemical calculation. Thereby, we obtain an accurate description of the active site in all the different protonation and oxidation states, and we can determine which of the 12 structures fit the experimental data best by comparing the crystallographic R-factors, electron-density maps, strain energies, and deviation from the ideal structure. The results indicate that FeIII OH- and FeIV OH- fit the experimental data almost equally well. These two states are appreciably better than the standard model of compound II, FeIV O2-. Combined with the available spectroscopic data, this indicates that compound II in myoglobin is protonated and is best described as FeIV OH-. It accepts a hydrogen bond from the distal His, which may be protonated at low pH.

Binding Sites↗

PubMed2004

How O2 binds to heme: reasons for rapid binding and spin inversion.

We have used density functional methods to calculate fully relaxed potential energy curves of the seven lowest electronic states during the binding of O(2) to a realistic model of ferrous deoxyheme. Beyond a Fe-O distance of approximately 2.5 A, we find a broad crossing region with five electronic states within 15 kJ/mol. The almost parallel surfaces strongly facilitate spin inversion, which is necessary in the reaction of O(2) with heme (deoxyheme is a quintet and O(2) a triplet, whereas oxyheme is a singlet). Thus, despite a small spin-orbit coupling in heme, the transition probability approaches unity. Using reasonable parameters, we estimate a transition probability of 0.06-1, which is at least 15 times larger than for the nonbiological Fe-O(+) system. Spin crossing is anticipated between the singlet ground state of bound oxyheme, the triplet and septet dissociation states, and a quintet intermediate state. The fact that the quintet state is close in energy to the dissociation couple is of biological importance, because it explains how both spin states of O(2) may bind to heme, thereby increasing the overall efficiency of oxygen binding. The activation barrier is estimated to be <15 kJ/mol based on our results and Mössbauer experiments. Our results indicate that both the activation energy and the spin-transition probability are tuned by the porphyrin as well as by the choice of the proximal heme ligand, which is a histidine in the globins. Together, they may accelerate O(2) binding to iron by approximately 10(11) compared with the Fe-O(+) system. A similar near degeneracy between spin states is observed in a ferric deoxyheme model with the histidine ligand hydrogen bonded to a carboxylate group, i.e. a model of heme peroxidases, which bind H(2)O(2) in this oxidation state.

Electrons↗

PubMed2004

On the role of the axial ligand in heme proteins: a theoretical study.

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys<Tyr<Tyr+Arg<His+Asp<His is found, reflecting the donor capacity of the various ligands. Thus, it is easier to reduce compound I with a His+Asp ligand than with a Cys ligand, in accordance with the one-electron chemistry of peroxidases and the hydroxylation reactions of cytochromes P450. However, the Tyr complexes have an unusually low affinity for all neutral ligands, giving them a slightly enhanced driving force in the oxidation of H(2)O(2) by compound I.

Amino Acid Substitution↗

PubMed2004

The structure of sitting-atop complexes of metalloporphyrins studied by theoretical methods.

The metallation of tetrapyrroles is believed to proceed via a sitting-atop (SAT) complex, in which some of the pyrrole nitrogen atoms are still protonated and the metal ion resides above the ring plane. No crystal structure of such a complex has been presented, but NMR and extended X-ray absorption fine structure (EXAFS) data has been reported for Cu(2+) in acetonitrile. We have used density functional calculations to obtain reasonable models for SAT complexes of porphyrins with Mg(2+), Fe(2+), and Cu(2+). The results show that there are many possible SAT complexes with 1-5 solvent molecules, one or two metal ions, and cis or trans protonation of the porphyrin ring. Many of these have similar energies and their relative stabilities vary with the metal ion. A complex with two cis pyrrolenine nitrogens atoms and 2-4 solvent molecules coordinated to Cu(2+) fits the NMR and EXAFS data best. However, we cannot fully exclude the possibility that what is observed is rather a mixture of a doubly protonated porphyrin and the copper porphyrin. Mg(2+) has a lower affinity for porphyrin and stronger affinity for water, so a complex with five water molecules and only one bond to porphyrin seems to be most stable. For Fe(2+), a cis structure with two first-sphere water molecules and four interactions to the porphyrin seems to be most likely.

Journal Article↗

PubMed2004

Protonation status of metal-bound ligands can be determined by quantum refinement.

The protonation status of key residues and bound ligands are often important for the function of a protein. Unfortunately, protons are not discerned in normal protein crystal structures, so their positions have to be determined by more indirect methods. We show that the recently developed quantum refinement method can be used to determine the position of protons in crystal structures. By replacing the molecular-mechanics potential, normally used in crystallographic refinement, by more accurate quantum chemical calculations, we get information about the ideal structure of a certain protonation state. By comparing the refined structures of different protonation states, the one that fits the crystallographic raw data best can be decided using four criteria: the R factors, electron density maps, strain energy, and divergence from the unrestrained quantum chemical structure. We test this method on alcohol dehydrogenase, for which the pK(a) of the zinc-bound solvent molecule is experimentally known. We show that we can predict the correct protonation state for both a deprotonated alcohol and a neutral water molecule.

Alcohol Dehydrogenase↗

PubMed2003

Quantum chemistry can locally improve protein crystal structures.

We have re-refined the X-ray structure of the heme site in cytochrome c553, supplementing the crystallographic data with quantum chemical geometry optimizations, instead of the molecular mechanics force field used in standard crystallographic refinement. By comparing the resulting structure, obtained using medium-resolution data (170 pm), with an atomic-resolution structure (95 pm) of the same protein, we show that the inclusion of quantum chemical information into the refinement procedure improves the structure significantly. Thus, errors in the Fe-ligand distances are reduced from 3 to 32 pm in the low-resolution structure to 0-5 pm in the re-refined structure, one side-chain atom changes its conformation (a movement by 214 pm toward its position in the high-resolution structure), and the R factors are improved by up to 0.018. Thus, quantum refinement may be a powerful method to obtain an accurate structure for interesting parts of a protein.

Crystallography, X-Ray↗

PubMed2003

Conversion of homocysteine to methionine by methionine synthase: a density functional study.

This communication reports a theoretical study of the conversion of homocysteine to methionine by methionine synthase. The reaction pathway is based on density functional calculations with large basis sets, including thermodynamic, relativistic, and solvent effects. We find that the suggested SN2 mechanism explains well the experimentally observed reaction rate. The results show that the reaction is highly polar, as reflected in the change of charge density along the reaction coordinate. It is enhanced in the protein by two effects: deprotonation of the bound substrate and desolvation of substrate and cofactor in the rate-determining step.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2003

Comparison of the chemical properties of iron and cobalt porphyrins and corrins.

Density functional calculations have been used to compare various geometric, electronic and functional properties of iron and cobalt porphyrin (Por) and corrin (Cor) species. The investigation is focussed on octahedral M(II/III) complexes (where M is the metal) with two axial imidazole ligands (as a model of b and c type cytochromes) or with one imidazole and one methyl ligand (as a model of methylcobalamin). However, we have also studied some five-coordinate M(II) complexes with an imidazole ligand and four-coordinate M(I/II) complexes without any axial ligands as models of other intermediates in the reaction cycle of coenzyme B12. The central cavity of the corrin ring is smaller than that of porphine. We show that the cavity of corrin is close to ideal for low-spin Co(III), Co(II), and Co(I) with the axial ligands encountered in biology, whereas the cavity in porphine is better suited for intermediate-spin states. Therefore, the low-spin state of Co is strongly favoured in complexes with corrins, whereas there is a small energy difference between the various spin states in iron porphyrin species. There are no clear differences for the reduction potentials of the octahedral complexes, but [Co(I)Cor] is more easily formed (by at least 40 kJ mole(-1)) than [Fe(I)Por]. Cobalt and corrin form a strong Cobond;C bond that is more stable against hydrolysis than iron and porphine. Finally, Fe(II/III) gives a much lower reorganisation energy than Co(II/III); this is owing to the occupied d(z2) orbital in Co(II). Altogether, these results give some clues about how nature has chosen the tetrapyrrole rings and their central metal ion.

Cobalt↗

PubMed2003

Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor.

The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu(2+), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with the carboxylate group of Asp(262). It is suggested that this occurs through a strong concomitant interaction of one of the oxygen's directly with the metal ion and the other oxygen to one of the nitrogens of the cyclam ring through a hydrogen bond.

Amino Acid Sequence↗

PubMed2003

An automatic method to generate force-field parameters for hetero-compounds.

A program, Hess2FF, has been developed that automatically constructs parameter and topology files to be used in crystallographic refinement for any molecule, based on a Hessian (force-constant) matrix estimated by any method. The program is tested by redefining hetero-compounds in five different proteins: the inhibitor N-methylmesoporphyrin bound to ferrochelatase, the haem group and its axial ligands in cyctochrome c(553), the active-site metal ion in iron superoxide dismutase, the catalytic zinc ion in alcohol dehydrogenase with a bound trifluoroethanol molecule and the 5'-deoxyadenosyl group in methylmalonyl coenzyme A mutase. It is shown that the resulting structures are improved in several aspects. In particular, the free R(free) factor always decreases and it is shown that a 1.70 A structure of cyctochrome c(553) becomes more similar to a high-resolution (0.97 A) structure of the same protein after re-refinement with Hess2FF. Thus, the force field used in crystallographic refinement significantly affects the final structure and therefore should be published together with the structure to ensure reproducibility. Various methods of obtaining the Hessian matrix employed by Hess2FF are discussed and some recommendations are given. Hess2FF allows the user to divide the atoms of the molecule into atom types that share the same force-field parameters. However, it seems to be favourable to assign a separate type to each atom, which can be performed automatically.

Algorithms↗

PubMed2003

Metal binding to Bacillus subtilis ferrochelatase and interaction between metal sites.

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn(2+), which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd(2+), an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg(2+), which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg(2+) has a stimulatory effect on the enzyme, lowering K(M) for Zn(2+) from 55 to 24 micro M. Changing one of the Mg(2+) binding residues, Glu272, to serine abolishes the effect of Mg(2+). It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg(2+) site may stimulate metal release from the protein ligands and its insertion into the porphyrin.

Bacillus subtilis↗

PubMed2003

Combined quantum and molecular mechanics calculations on metalloproteins.

The combination of quantum mechanics and molecular mechanics (QM/MM) methods is one of the most promising approaches to study the structure, function and properties of proteins. The number of QM/MM applications on metalloproteins is steadily increasing, especially studies with density functional methods on redox-active metal centres. Recent developments include new parameterised methods to treat covalent bonds between the quantum and classical systems, methods to obtain free energy from QM/MM results, and the combination of quantum chemistry and protein crystallography.

Crystallography, X-Ray↗

PubMed2002

The importance of porphyrin distortions for the ferrochelatase reaction.

Ferrochelatase is the terminal enzyme in haem biosynthesis, i.e. the enzyme that inserts a ferrous ion into the porphyrin ring. Suggested reaction mechanisms for this enzyme involve a distortion of the porphyrin ring when it is bound to the enzyme. We have examined the energetics of such distortions using various theoretical calculations. With the density functional B3LYP method we calculate how much energy it costs to tilt one of the pyrrole rings out of the porphyrin plane for an isolated porphyrin molecule without or with a divalent metal ion in the centre of the ring. A tilt of 30 degrees costs 65-130 kJ/mol for most metal ions, but only approximately 48 kJ/mol for free-base (neutral) porphine. This indicates that once the metal is inserted, the porphyrin becomes stiffer and flatter, and therefore binds with lower affinity to a site designed to bind a distorted porphyrin. This would facilitate the release of the product from ferrochelatase. This proposal is strengthened by the fact that the only tested metal ion with a lower distortion energy than free-base porphyrin (Cd(2+)) is an inhibitor of ferrochelatase. Moreover, it costs even less energy to tilt a doubly deprotonated porphine(2-) molecule. This suggests that the protein may lower the acid constant of the pyrrole nitrogen atoms by deforming the porphyrin molecule. We have also estimated the structure of the protoporphyrin IX substrate bound to ferrochelatase using combined quantum chemical and molecular mechanics calculations. The result shows that the protein may distort the porphyrin by approximately 20 kJ/mol, leading to a distinctly non-planar structure. All four pyrrole rings are tilted out of the porphyrin mean plane (1-16 degrees ) but most towards the putative binding site of the metal ion. The predicted tilt is considerably smaller than that observed in the crystal structure of a porphyrin inhibitor.

Binding Sites↗

PubMed2002

Theoretical study of the discrimination between O(2) and CO by myoglobin.

Combined quantum chemical and molecular mechanics geometry optimisations have been performed on myoglobin without or with O(2) or CO bound to the haem group. The results show that the distal histidine residue is protonated on the N(epsilon 2) atom and forms a hydrogen bond to the haem ligand both in the O(2) and the CO complexes. We have also re-refined the crystal structure of CO[bond]myoglobin by a combined quantum chemical and crystallographic refinement. Thereby, we probably obtain the most accurate available structure of the active site of this complex, showing a Fe[bond]C[bond]O angle of 171 degrees, and Fe[bond]C and C[bond]O bond lengths of 170-171 and 116-117 pm. The resulting structures have been used to calculate the strength of the hydrogen bond between the distal histidine residue and O(2) or CO in the protein. This amounts to 31-33 kJ/mol for O(2) and 2-3 kJ/mol for CO. The difference in hydrogen-bond strength is 21-22 kJ/mol when corrected for entropy effects. This is slightly larger than the observed discrimination between O(2) or CO by myoglobin, 17 kJ/mol. We have also estimated the strain of the active site inside the protein. It is 2-4 kJ/mol larger for the O(2) complex than for the CO complex, independent of which crystal structure the calculations are based on. Together, these results clearly show that myoglobin discriminates between O(2) and CO mainly by electrostatic interactions, rather than by steric strain.

Binding Sites↗

PubMed2002

Restrained point-charge models for disaccharides.

Various methods for deriving atomic partial charges from the quantum chemical electrostatic potential and moments have been tested for the sucrose molecule. We show that if no further information is used, the charges on some carbon atoms become large and charge patterns involving these atoms are badly determined and poorly transferable. Adding lone-pairs on the ether oxygen atoms or dividing the molecule into smaller fragments did not cure the instabilities. We develop a method, CHELP-BOW0, that restrains charges toward zero with different weights for different atoms. These harmonic restraints preserve the linear form of the least-squares equations, which are solved in a single step using singular-value decomposition. CHELP-BOW0 improves the chemical transferability of the charges compared to unrestrained methods, and slightly improves their conformational transferability. It introduces a modest degradation of the fit compared to unrestrained CHELP-BOW (mean average deviation of the potential 0.00016 vs. 0.00010 a.u.). A second new method, CHELP-BOWC, avoids the need for restraints by including several conformations in the fit, weighting each according to its estimated energy in solution. CHELP-BOWC charges are more transferable than CHELP-BOW or CHELP-BOW0 charges to conformations not included in the training set. Restraints to zero charge do not further improve transferability of the CHELP-BOWC charges. We, therefore, recommend CHELP-BOW charges for rigid molecules and CHELP-BOWC charges for flexible molecules.

Computer Simulation↗

PubMed2002

Quantum chemical geometry optimizations in proteins using crystallographic raw data.

A method is developed for the combination of quantum chemical geometry optimizations and crystallographic structure refinement. The method is implemented by integrating the quantum chemical software Turbomole with the crystallographic software Crystallography and NMR System (CNS), using three small procedures transferring information between the two programs. The program (COMQUM-X)is used to study the binding of the inhibitor N-methylmesoporphyrin to ferrochelatase, and we show that the method behaves properly and leads to an improvement of the structure of the inhibitor. It allows us to directly quantify in energy terms how much the protein distort the structure of the bound inhibitor compared to the optimum vacuum structure (4-6 kJ/mol). The approach improves the standard combined quantum chemical and molecular mechanics (QC/MM) approach by guaranteeing that the final structure is in accordance with experimental data (the reflections) and avoiding the risk of propagating errors in the crystal coordinates. The program can also be seen as an improvement of standard crystallographic refinement, providing an accurate empirical potential function for any group of interest. The results can be directly interpreted in standard crystallographic terms (e.g., R factors or electron density maps). The method can be used to interpret crystal structures (e.g., the protonation status of metal-bound water molecules) and even to locally improve them.

Binding Sites↗