Moderating influence of proteins on nonplanar tetrapyrrole deformations: coenzyme F430 in methyl-coenzyme-M reductase

Normal-coordinate structural decomposition, cluster analysis, and molecular mechanics calculations were undertaken to examine the effect of methyl-coenzyme-M reductase (MCR) on the nonplanar deformations of coenzyme F430. Although free 12,13-diepi-F430 has a lower energy conformation than free F430, the protein restraints exerted by MCR are responsible for F430 having a lower energy conformation than the 12,13-diepimer in MCR. According to the NSD analysis, the crystal structure of free diepimerized F430M is highly distorted. In MCR the protein prevents 12,13-diepi-F430 from undergoing nonplanar deformations; therefore, MCR favors F430 over the 12,13-diepimeric form. The strain imposed on 12,13-diepi-F430 in the protein is so large that although 88% of free F430 is found in the diepimeric form, none of the diepimeric form is found in MCR. This is of significance since the two forms have different chemistries. MCR also moderates the nonplanar deformations of coenzyme F430, which are known to affect redox potentials and axial ligand affinities in tetrapyrroles, suggesting that the protein environment (MCR) is responsible for tuning the chemistry of the active site nickel ion. F430 is bound to MCR by hydrogen bonds between the protein and the F430 carboxylate groups. Conformational searches have shown that F430 has very little rotational and translational freedom within MCR.     

Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.     

The nickel-sirohydrochlorin formation mechanism of the ancestral class II chelatase CfbA in coenzyme F430 biosynthesis

The class II chelatase CfbA catalyzes Ni²⁺ insertion into sirohydrochlorin (SHC) to yield the product nickel-sirohydrochlorin (Ni-SHC) during coenzyme F430 biosynthesis. CfbA is an important ancestor of all the class II chelatase family of enzymes, including SirB and CbiK/CbiX, functioning not only as a nickel-chelatase, but also as a cobalt-chelatase in vitro. Thus, CfbA is a key enzyme in terms of diversity and evolution of the chelatases catalyzing formation of metal-SHC-type of cofactors. However, the reaction mechanism of CfbA with Ni²⁺ and Co²⁺ remains elusive. To understand the structural basis of the underlying mechanisms and evolutionary aspects of the class II chelatases, X-ray crystal structures of Methanocaldococcus jannaschii wild-type CfbA with various ligands, including SHC, Ni²⁺, Ni-SHC, and Co²⁺ were determined. Further, X-ray crystallographic snapshot analysis captured a unique Ni²⁺-SHC-His intermediate complex and Co-SHC-bound CfbA, which resulted from a more rapid chelatase reaction for Co²⁺ than Ni²⁺. Meanwhile, an in vitro activity assay confirmed the different reaction rates for Ni²⁺ and Co²⁺ by CfbA. Based on these structural and functional analyses, the following substrate-SHC-assisted Ni²⁺ insertion catalytic mechanism was proposed: Ni²⁺ insertion to SHC is promoted by the support of an acetate side chain of SHC.

The substrate-assisted nickel chelatase mechanism of CfbA in coenzyme F430 biosynthesis was unveiled by X-ray crystal structure analysis.     

Nickel oxidation states of F(430) cofactor in methyl-coenzyme M reductase

Magnetic circular dichroism (MCD) spectroscopy and variable-temperature variable-field MCD are used in combination with density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations to characterize the so-called ox1-silent, red1, and ox1 forms of the Ni-containing cofactor F430 in methyl-coenzyme M reductase (MCR). Previous studies concluded that the ox1 state, which is the precursor of the key reactive red1 state of MCR, is a Ni(I) species that derives from one-electron reduction of the Ni(II)-containing ox1-silent state. However, our absorption and MCD data provide compelling evidence that ox1 is actually a Ni(II) species. In support of this proposal, our DFT and TD-DFT calculations indicate that addition of an electron to the ox1-silent state leads to formation of a hydrocorphin anion radical rather than a Ni(I) center. These results and biochemical evidence suggest that ox1 is more oxidized than red1, which prompted us to test a new model for ox1 in which the ox1-silent species is oxidized by one electron to form a thiyl radical derived from coenzyme M that couples antiferromagnetically to the Ni(II) ion. This alternative ox1 model, formally corresponding to a Ni(III)/thiolate resonance form but with predicted S = 1/2 EPR parameters reminiscent of a Ni(I) (3dx2-y2)1 species, rationalizes the requirement for reduction of ox1 to yield the red1 species and the seemingly incongruent EPR and electronic spectra of the ox1 state.     

Local deformations of polymers with nonplanar rigid main‐chain internal coordinates

A procedure for local deformation of a polymer by concerted rotation of several main‐chain dihedral angles has been adapted recently to be an elementary move in Monte Carlo simulations. We expand the applicability of the move by generalizing the formalism to allow fixed dihedral angles that sequentially interrupt the rotatable bonds to be nonplanar. The method is applied to the simulation of a small protein in which the dihedral angles of the peptide bonds are allowed to deviate from their ideal values and to the simulation of an RNA hairpin loop in which the main chain (C3′ C4′) bonds that are constrained by the sugar rings are rigid but nonplanar. The move is found to increase the rate at which the systems explore their accessible configuration spaces. The relation of the results to previous studies and possible enhancements of the method are discussed. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1132–1144, 2000     

Coenzyme F430 from Methanogenic Bacteria: Mechanistic studies on the reductive cleavage of sulfonium ions catalyzed by F430 pentamethyl ester

Mechanistic questions regarding the reductive cleavage of sulfonium ions by the Ni^I form of coenzyme F430 pentamethyl ester (F430M) were addressed in a series of kinetic studies and isotope labeling experiments. In neat DMF, methane formation from dialkyl(methyl)sulfonium ions consistently showed a delay time of ca. 1 h. In the presence of excess propanethiol, no delay was observed and methane formation followed pseudo-first-order kinetics with a logarithmic dependence of the initial rate on the concentration of propanethiol. From the temperature dependence of the reaction rate, an estimate for the activation parameters of ΔH^# = 49 kJ mol^?1 and (apparent) ΔS# = –114 J K^?1 mol^?1 was derived. The observation of deuterium incorporation into methane from (CH₃)₂CHOD, but not from (CH₃)₂CDOH, indicates that the fourth H-entity is introduced into CH₄ as a proton, and that free CH₃ radicals are not involved. In contrast to the reaction with the homogeneous one-electron reductant sodium naphthalide, the F430M-catalyzed reduction of mixed dialkyl(methyl)sulfonium ions showed a pronounced selectivity for the cleavage of Me? S over that of alkyl-S (alkyl ≠ Me) bonds. Mechanisms that are consistent with these results, as well as possible explanations for the time delay and the apparent highly negative entropy of activation, are discussed.     

Coenzyme F430 from Methanogenic Bacteria: Oxidation of F430 Pentamethyl Ester to the Ni(III) Form

F430M, the pentamethyl ester of coenzyme F430, can be oxidized reversibly by one electron. The oxidation potential has been determined, and the electrolytically prepared oxidation product was characterized by its UV/VIS and ESR spectrum. The strongly anisotropic and nearly axial ESR spectrum is consistent with a S = ½ species with the unpaired-electron spin density predominantly in a d-type orbital of the central nickel ion. The properties of Ni(III)F430M are discussed in the context of two hypothetical mechanisms for the catalytic role of coenzyme F430 in methyl coenzyme M reductase, which catalyses the last step of methane formation in methanogenic bacteria.     

Coordination of methyl coenzyme M and coenzyme M at divalent and trivalent nickel cyclams: model studies of methyl coenzyme M reductase active site

Divalent and trivalent nickel complexes of 1,4,8,11-tetraazacyclotetradecane, denoted as cyclam hereafter, coordinated by methyl coenzyme M (MeSCoM(-)) and coenzyme M (HSCoM(-)) have been synthesized in the course our model studies of methyl coenzyme M reductase (MCR). The divalent nickel complexes Ni(cyclam)(RSCoM)(2) (R = Me, H) have two trans-disposed RSCoM(-) ligands at the nickel(II) center as sulfonates, and thus, the nickels have an octahedral coordination. The SCoM(2-) adduct Ni(cyclam)(SCoM) was also synthesized, in which the SCoM(2-) ligand chelates the nickel via the thiolate sulfur and a sulfonate oxygen. The trivalent MeSCoM adduct [Ni(cyclam)(MeSCoM)(2)](OTf) was synthesized by treatment of [Ni(cyclam)(NCCH(3))(2)](OTf)(3) with ((n)Bu(4)N)[MeSCoM]. A similar reaction with ((n)Bu(4)N)[HSCoM] did not afford the corresponding trivalent HSCoM(-) adduct, but rather the divalent nickel complex polymer [-Ni(II)(cyclam)(CoMSSCoM)-](n) was obtained, in which the terminal thiol of HSCoM(-) was oxidized to the disulfide (CoMSSCoM)(2-) by the Ni(III) center.     

Direct determination of the number of electrons needed to reduce coenzyme F430 pentamethyl ester to the Ni(I) species exhibiting the electron paramagnetic resonance and ultraviolet-visible spectra characteristic for the MCR(red1) state of methyl-coenzyme M reductase

The UV-visible and electron paramagnetic resonance (EPR) spectra of MCR(red1), the catalytically active state of methyl-coenzyme M reductase, are almost identical to those observed when free coenzyme F430 or its pentamethyl ester (F430M) are reduced to the Ni(I) valence state. Investigations and proposals concerning the catalytic mechanism of MCR were therefore based on MCR(red1) containing Ni(I)F430 until, in a recent report, Tang et al. (J. Am. Chem. Soc. 2002, 124, 13242) interpreted their resonance Raman data and titration experiments as indicating that, in MCR(red1), coenzyme F430 is not only reduced at the nickel center but at one of the C=N double bonds of the hydrocorphinoid macrocycle as well. To resolve this contradiction, we have investigated the stoichiometry of the reduction of coenzyme F430 pentamethyl ester (F430M) by three independent methods. Spectroelectrochemistry showed clean reduction to a single product that exhibits the UV-vis spectrum typical for MCR(red1). In three bulk electrolysis experiments, 0.96 +/- 0.1 F/mol was required to generate the reduced species. Reduction with decamethylcobaltocene in tetrahydrofuran (THF) consumed 1 mol of (Cp)(2)Co/mol of F430M, and the stoichiometry of the reoxidation of the reduced form with the two-electron oxidant methylene blue was 0.46 +/- 0.05 mol of methylene blue/mol of reduced F430M. These experiments demonstrate that the reduction of coenzyme F430M to the species having almost identical UV-vis and EPR spectra as MCR(red1) is a one-electron process and therefore inconsistent with a reduction of the macrocycle chromophore.     

Effect of contact deformations on the adhesion of particles

A strict theory of reciprocal influence of the contact deformation and molecular attraction of a ball and a plane has been developed. It has been shown that despite the van der Waals' forces being capable of increasing the elastic contact area between the ball and the plane, the force that is required to overcome the molecular forces arising when the contact is broken does not increase thereby. In fact, it remains equal to the attraction force value that is determined when considering the point contact of a nondeformed ball with a plane.

In the absence of the electrostatic component, the adhesion force is equivalent to the first power of the ball radius and to the amount of work per unit area as required for affecting the equilibrium tearing-off of a flat surface of the same nature.     

Coenzyme F430 from Methanogenic Bacteria: Complete Assignment of Configuration Based on an X-Ray Analysis of 12,13-Diepi-F430 Pentamethyl Ester and on NMR Spectroscopy

The structure of a derivative of coenzyme F430 from methanogenic bacteria, the bromide salt of 12,13-diepi-F430 pentamethyl ester ( 5 , X = Br), was determined by X-ray structure analysis. It reveals a more pronounced saddle-shaped out-of-plane deformation of the macrocycle than any hydroporphinoid Ni complex investigated so far. The crystal structure confirms the constitution proposed for coenzyme F430 ( 2 ) and shows that in the epimer 5 , the three stereogenic centers in ring D, C(17), C(18), and C(19), have the (17S)-, (18S)-, and (19R)-configuration, respectively. Deuteration and 2D-NMR studies independently demonstrate that native coenzyme F430 (2) has the same configuration in ring D as the epimer 5 . Therefore, our original tentative assignment of configuration at C(19) and C(18) [1] has to be reversed. This completes the assignment of configuration for all stereogenic centers in coenzyme F430, which has the structure shown in Formula 2 .     

Application of MCD spectroscopy and TD-DFT to nonplanar core-modified tetrabenzoporphyrins: effect of reduced symmetry on nonplanar porphyrinoids

The optical spectra of a series of core-modified tetrabenzoporphyrins were analyzed to determine the effects of core modification, ligand folding, and partial benzo substitution at the ligand periphery on the electronic structure by using magnetic circular dichroism (MCD) and NMR spectroscopy, X-ray crystallography, cyclic and differential pulse voltammetry, and TD-DFT calculations. Planar 21-carba-, 21-thia-, 21,23-dithia-, and 21-oxa-23-thiatetrabenzo[b,g,l,q]porphyrins reported previously were studied together with the previously unreported 21-oxa- and 21-carba-23-thiatetrabenzo[b,g,l,q]porphyrins. The optical properties of these compounds are compared to those of tetrabenzo[b,g,l,q]-, 5,10,15,20-tetraphenyl-, 5,10,15,20-tetraphenyltetrabenzo[b,g,l,q]-21-thia-, 5,10,15,20-tetraphenyltetrabenzodithia-, 5,10,15,20-tetraphenyldibenzo[g,q]-21,23-dithia-, 5,10,15,20-tetraphenyldibenzo[b,l]-21,23-dithia-, 5,10,15,20-tetraphenyltribenzo[g,q,l]-21-thia-, and 5,10,15,20-tetraphenylbenzo[b]-21-thiaporphyrins. Michl's perimeter model and Gouterman's four-orbital model are used to conceptualize the results and to account for red shifts commonly observed in the spectral bands of nonplanar porphyrinoids.     

Novel topological index F based on incidence matrix

Based on incidence matrix W, the novel topological index F is defined by the matrices L, W, X as F = LWX. The properties, the chemical environments, and the interaction of the vertexes in a molecule are taken into account in this definition. Several good QSPR models in the hetero-atom-containing organic compounds and inorganic compounds are obtained. Moreover, based on the definition of F and the values C(i) of the vertexes or the values (m)F(ij) of the chemical bonds, we have obtained serial indices, (m)F(v), (m)F(b), and F(w). They are successfully applied to QSPR models and good correlation results have been obtained as well.     

Planar and nonplanar vibrations of cytosine

The formalism developed in the preceding article is used in a normal coordinate analysis of the pyrimidine base cytosine. The results of both planar and nonplanar vibrations are reported. The model yields 66 frequencies with an average error of about 6.5 cm^?1 (ca. 0.5%). The vibrational modes are compared with experimental data and discussed in terms of potential energy distributions and Cartesian displacements. Two isolated low-frequency (near 200 cm^?1) out-of-plane modes are predicted in the vicinity of where two such modes are believed to occur. In addition, the model has taken into account the observed coalescence of the torsional and wagging modes of the amino group upon deuteration of the amino group. Recent data from sulfur-substituted cytosine (2-thiocytosine) were useful in making assignments.     

Rotation and alignment of anisotropic particles on nonplanar interfaces

We study the alignment of micron-scale particles at air-water interfaces with unequal principle radii of curvature by optical microscopy. The fluid interface bends to satisfy the wetting conditions at the three phase contact line where the interface intersects the particle, creating deflections that increase the area of the interface. These deflections decay far from the particle. The far field interface shape has differing principle radii of curvature over length scales large compared to the particle. The deflections create excess area which depends on the angle of the particle with respect to the principle axes of the interface. We show that when particles create surface deflections with quadrupolar modes, the particles rotate to preferred orientations to minimize the free energy. In experiment, we focus on uniform surface energy particles, for which quadrupolar modes are forced by the particle shape. Analytical expressions for the torque and stable states are derived in agreement with experiment and confirmed computationally.     

NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii

The 13C NMR resonance and one-bond 1H-13C coupling constants of coenzyme B12 enriched in 13C in the cobalt-bound carbon have been observed in the complex of the coenzyme with the B12-dependent ribonucleotide reductase from Lactobacillus leichmannii. Neither the 13C NMR chemical shift nor the 1H-13C coupling constants are significantly altered by binding of the coenzyme to the enzyme. The results suggest that ground-state Co-C bond distortion is not utilized by this enzyme to activate coenzyme B12 for C-Co bond homolysis.     

Effect of weak anchoring on the electric field induced deformations of nematic layers

The stationary deformations of nematic layers with a twisted structure are analysed by means of the Taylor expansion method based on catastrophe theory. The role of weak anchoring is investigated. Variations of the polar and azimuthal angles describing the surface director orientation are allowed. The stability of two equilibrium states, the twisted and the homeotropic, is studied. Several types of continuous and discontinuous transitions between them are revealed. The threshold voltages are calculated.     

Effect of coenzyme Q(10) incorporation on the characteristics of nanoliposomes

Coenzyme Q(10) (CoQ(10)) is incorporated in nanoliposomes composed of egg yolk phospholipid, cholesterol, and Tween 80. Atomic force microscopy, performed to characterize vesicle surface topology, shows some visible influence of CoQ(10) on the nanoliposomal structure. CoQ(10) incorporation can suppress the increase of the z-average diameter of nanoliposomes during storage for 8 months at 4 degrees C. The liposomal lipid peroxidation caused by Fe(III)/ascorbate is also significantly inhibited. Perturbation of acyl chain motion of lipids due to the presence of CoQ(10) in the bilayer is examined by fluorescence probe diphenyl-hexatriene and Raman spectroscopy. Fluorescence probe studies indicate that CoQ(10) incorporation results in the microviscosity increase of nanoliposomes. The steric structure of nanoliposomes reflected by Raman spectroscopy changes obviously and shows CoQ(10) content dependency. The order parameters for the lateral interaction between chains increase. The trans conformation decrease and the gauche conformation increase as the weight contents of CoQ(10) incorporation are at 1%, 5%, 10%, and 32.5%. However, the order parameters for the longitudinal interaction in chains was higher than that of pure nanoliposomes as the weight content of CoQ(10) is at 25%. Results suggest that CoQ(10)might intercalate between lipid molecules and perturb the bilayer structure.     

Immobilization ofmyo-inositol-1-phosphate synthase containing active,self-regenerating coenzyme (NAD+) on the same matrix

myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testes, an NAD⁺-containing enzyme, which convertsd-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, could be immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde at pH 4.5. The enzyme bound to the gel showed a specific activity of 5.6% of that of the native enzyme, but the activity could be increased to 21% by pretreatment with urea.     

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.