Applications of the Newly Developed Force-Field Parameters Uncover a Dynamic Nature of Ω-Loop C in the Lys-Ligated Alkaline Form of Cytochrome c.

Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso-1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.

Tryptophan Stabilization of a Biochemical Carbocation Evaluated by Analysis of π Complexes of 3-Ethylindole with the t-Butyl Cation.

Understanding how the highly unstable carbocation intermediates in terpenoid biosynthesis are stabilized and protected during their transient existence in enzyme active sites is an intriguing challenge which has to be addressed computationally. Our efforts have focused on evaluating the stabilization afforded via carbocation-π complexation between a biochemical carbocation and an aromatic amino acid residue. This has involved making measurements on an X-ray structure of an enzyme active site that shows a π donor proximate to a putative carbocation site and using these to build models which are analyzed computationally to provide an estimated stabilization energy (SE). Previously, we reported estimated SEs for several such carbocation-π complexes involving phenylalanine. Herein, we report the first such estimate involving tryptophan as the π donor. Because there was almost no published information about indole as a π-complexation donor, we first located computationally equilibrium π and σ complexes of 3-ethylindole with the t-butyl cation as relevant background information. Then, measurements on the X-ray structure of the enzyme CotB2 complexed with geranylgeranyl thiodiphosphate (GGSPP), specifically on the geometric relationship of the putative carbocation at C15 of GGSPP to W186, were used to build a model that afforded a computed SE of -15.3 kcal/mol.

A simpler method affords evaluation of π stabilization by phenylalanine of several biochemical carbocations.

Carbocations are important intermediates in the biosynthesis of terpenes and steroids, and it is challenging to try to understand how these relatively unstable species survive even transiently during biochemical reactions. Carbocation-π interaction with aromatic amino acid residues is an important factor in helping to stabilize these positively charged species. However, the short lifetimes of these active site carbocations makes experimental evaluation of the stabilization afforded by such interaction impossible. Computational studies, however, have provided some insight into this phenomenon. Herein we report a simple, computationally efficient method to estimate such stabilization energies afforded by phenylalanine to biochemical carbocation intermediates. A model is constructed in which the biochemical carbocation is replaced by an appropriate carbocation mimic (t-butyl or dimethylallyl). This substitute carbocation is then aligned with an ethylbenzene serving as a surrogate for each proximate phenylalanine in a geometry that replicates as closely as possible the orientation of that phenylalanine using measurements made on an X-ray structure of an enzyme active site in which a carbocation surrogate is bound. Density functional theory computations on such models were then used to yield estimates of stabilization energies. Application of this method to the tertiary carbocation formed in the reaction catalyzed by geranyl diphosphate C-methyl transferase gave a stabilization energy (-12.3 kcal mol-1) that was essentially identical to that obtained previously by analysis of a much more computationally demanding model of the active site. As a check on the accuracy of the simpler method, it was applied with similar success to the farnesyl cation formed in the reaction catalyzed by aristolochene synthase that is stabilized by cation-π interaction with two phenylalanines. Application of this method is also described to estimate carbocation-π stabilization, by the same two phenylalanines, of the final carbocation intermediate leading to aristolochene through analysis of the X-ray structure of an inhibitor of that carbocation bound in the active site of aristolochene synthase. Finally, the stabilization, by either of two phenylalanines, of six carbocation intermediates in the oxidosqualene cyclase-catalyzed formation of lanosterol is estimated by comparable analysis of an X-ray structure of that reaction product bound in the enzyme active site.

Carbocation-π interaction: evaluation of the stabilization by phenylalanine of a biochemical carbocation intermediate.

Computational analyses, using primarily density functional theory, have been used to determine the stabilization associated with the carbocation-π interaction of a biochemical carbocation intermediate binding to a phenylalanine residue in an enzyme active site. Studies of complexation between t-butyl cation and ethylbenzene, and of a model of a carbocation intermediate with a phenylalanine in the active site of geranyl diphosphate C-methyl transferase, have afforded the first quantitative evaluation of the stabilization that can be provided to a carbocation by an aromatic residue in an enzymatic reaction. Describing the hydrophobic surrounding medium using a dielectric constant between ε = 2 and ε = 4, the calculated carbocation-π stabilization energy lies in the range of 10-7.5 kcal mol-1.

A comparison of stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex.

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.

Metal Complexes of Mesitylphosphine: Synthesis, Structure, and Spectroscopy.

A series of primary phosphine homoleptic complexes [ML(4)](n)()(+)X(n)() (1, M = Ni, n = 0; 2, M = Pd, n = 2, X = BF(4); 3, M = Cu, n = 1, X = PF(6); 4, M = Ag, n = 1, X = BF(4); L = PH(2)Mes, Mes = 2,4,6-Me(3)C(6)H(2)] was prepared from mesitylphosphine and Ni(COD)(2), [Pd(NCMe)(4)][BF(4)](2), [Cu(NCMe)(4)]PF(6), and AgBF(4), respectively. Reactions of 1-4 with MeC(CH(2)PPh(2))(3) (triphos) or [P(CH(2)CH(2)PPh(2))(3)] (tetraphos) afforded the derivatives [M(L')L](n)()(+)X(n)() (L' = triphos; 6, M = Ni, n = 0; 7, M = Cu, n = 1, X = PF(6); 8, M = Ag, n = 1, X = BF(4); L' = tetraphos; 9, M = Pd, n = 2, X = BF(4)). Addition of NOBF(4) to 1 yielded the nitrosyl compound [NiL(3)(NO)]BF(4), 5. The solution structure and dynamics of 1-9 were studied by (31)P NMR spectroscopy (including the first reported analyses of a 12-spin system for 1-2). Complexes 1, 3, 6, and 7.solvent were characterized crystallographically. The structural and spectroscopic studies suggest that the coordination properties of L are dominated by its relatively small cone angle and that the basicity of L is comparable to that of more commonly used tertiary phosphines.