A QM/MM study of the nature of the entatic state in plastocyanin

Plastocyanin is a copper containing protein that is involved in the electron transfer process in photosynthetic organisms. The active site of plastocyanin is described as an entatic state whereby its structure represents a compromise between the structures favored by the oxidized and reduced forms. In this study, the nature of the entatic state is investigated through density functional theory-based hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. The strain energy is computed to be 12.8 kcal/mol and 14.5 kcal/mol for the oxidized and reduced forms of the protein, indicating that the active site has an intermediate structure. It is shown that the energy gap between the oxidized and reduced forms varies significantly with the fluctuations in the structure of the active site at room temperature. An accurate determination of the reorganization energy requires averaging over conformation and a large region of the protein around the active site to be treated at the quantum mechanical level

A QM/MM study of the nature of the entatic state in plastocyanin

Plastocyanin is a copper containing protein that is involved in the electron transfer process in photosynthetic organisms. The active site of plastocyanin is described as an entatic state whereby its structure represents a compromise between the structures favored by the oxidized and reduced forms. In this study the nature of the entatic state is investigated through density functional theory based hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. The strain energy is computed to be 12.8 kcal/mol and 14.5 kcal/mol for the oxidized and reduced forms of the protein, indicating that the active site has an intermediate structure. It is shown that the energy gap between the oxidized and reduced forms varies significantly with the fluctuations in the structure of the active site at room temperature. An accurate determination of the reorganization energy requires averaging over conformation and a large region of the protein around the active site to be treated at the quantum mechanical level

Computational study of the structure and electronic circular dichroism spectroscopy of blue copper proteins

The calculation of the electronic circular dichroism (CD) spectra of the oxidised form of the blue copper proteins plastocyanin and cucumber basic protein and the relationship between the observed spectral features and the structure of the active site of the protein is investigated. Excitation energies and transition strengths are computed using multi reference configuration interaction, and it is shown that computed spectra based on coordinates from the crystal structure or a single structure optimised in quantum mechanics/molecular mechanics (QM/MM) or ligand field molecular mechanics (LFMM) are qualitatively incorrect. In particular, the rotational strength of the ligand to metal charge transfer band is predicted to be too small or have the incorrect sign. By considering calculations on active site models with modified structures it is shown that the intensity of this band is sensitive to the non-planarity of the histidine and cysteine ligands coordinated to copper. Calculation of the ultraviolet absorption and CD spectra based upon averaging over many structures drawn from a LFMM molecular dynamics simulation are in good agreement with experiment, and superior to analogous calculations based upon structures from a classical molecular dynamics simulation. This provides evidence that the LFMM force field provides an accurate description of the molecular dynamics of these proteins

Outer-Sphere Effects on the Copper Sites of Pseudomonas Aeruginosa Azurins

This thesis describes structural, spectroscopic, and reactivity studies of mutants of the cupredoxin azurin from Pseudomonas aeruginosa. Emphasis is given to the effects of outer-sphere coordination on spectroscopic observables and reduction potentials. Among the key findings are that the characteristics of the classical "type 1" copper site can be largely reproduced in the absence of canonically necessary cysteine thiolate ligation. Mutants exhibiting this behavior have been classified as "type zero" copper proteins.</p

Spectroscopic and DFT studies of second-sphere variants of the type 1 copper site in azurin: covalent and nonlocal electrostatic contributions to reduction potentials

The reduction potentials (E^0) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by ~400 mV. Here, we use a combination of low-temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and S K-edge X-ray absorption spectroscopies to investigate a series of second-sphere variants--F114P, N47S, and F114N in Pseudomonas aeruginosa azurin--which modulate hydrogen bonding to and protein-derived dipoles nearby the Cu-S(Cys) bond. Density functional theory calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E(0) into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active interactions, while affording further insight into the second-sphere mechanisms available to the protein to tune the E^0 of electron-transfer sites in biology

Regulatory mechanisms and manipulation of electron transfer properties in copper metalloproteins

En esta tesis se aborda el estudio de los parámetros que regulan las reacciones detransferencia electrónica (TE) en metaloproteínas que contienen dos tipos de sitios decobre distintos. Para ello se utilizó una variedad de métodos espectroscópicos yelectroquímicos, cuyos resultados fueron analizados en el marco de la teoría de Marcusde TE. El principal modelo de estudio es un fragmento soluble de la citocromo ba3oxidasa de Thermus thermophilus (Tt-CuA), que contiene el centro binuclear de CuA,aceptor de electrones primario de la enzima. Mediante distintas estrategias demutagénesis tales como mutaciones puntuales de primera esfera de coordinación oingeniería de bucles, se investigaron los factores que determinan las propiedadeselectrónicas del sitio nativo, y que en última instancia determinan los mecanismosregulatorios de la TE. Desde la óptica de la termodinámica estadística, se logróracionalizar las contribuciones entálpica y entrópica al potencial de reducción del sitiode CuA y se verificó la aditividad de los efectos producidos por las distintas mutacionesde primera y segunda esfera de coordinación. Diferentes estudios espectroscópicos (RR, RMN, UV-Vis, EPR) revelan que laestructura electrónica del sitio de CuA puede ser descripta mediante una superficie deenergía potencial con dos pozos que corresponden a estados fundamentalesalternativos. Más aún, la población relativa de estos estados puede ser modulada através de mutaciones de primera y segunda esfera, y en una mutante específicamediante el pH del medio. Empleando voltametría cíclica de films proteicos fue posibledeterminar los parámetros cinéticos de TE para ambos estados fundamentales demanera independiente. En particular, la mutante puntual de ligando axial M160Hconstituye el primer caso conocido en que resulta posible activar selectivamente dosestados fundamentales diferentes mediante un simple cambio de pH. Por otra parte, estudios realizados sobre sitios de cobre tipo 1 (T1) reconstruidos enel esqueleto proteico de Tt-CuA mediante ingeniería de bucles muestran clarasdiferencias en relación a los sitios T1 nativos. Grandes cambios espectroscópicos onotables desplazamientos de los potenciales redox demuestran el rol determinante dela matriz proteica sobre las propiedades estructurales y funcionales de los sitios redox. Estos sistemas novedosos presentan además la capacidad de modificar la coordinacióndel sitio activo a partir del agregado de un ligando exógeno sin perder su actividad redox,permitiendo el estudio de este tipo de fenómenos en un sistema biológico.The purpose of this thesis is to study the parameters that regulate electron transfer (ET) reactions in metalloproteins containing two different kinds of copper sites. A varietyof spectroscopic and electrochemical methods were used in the research and the resultswere analyzed in the context of Marcus ET theory. The main study model is a solublefragment of the cytochrome ba3 oxidase from Thermus thermophiles that contains the CuA binuclear center (Tt-CuA), which is the primary electron acceptor of this enzyme. Using different mutagenesis strategies, such as axial ligand point mutations -in the firstcoordination sphere- or loop engineering, elements defining the native site electronicproperties, which also define ET regulatory mechanisms, were analyzed. The entropicand enthalpic contributions to the reduction potential of the CuA site were rationalizedin terms of statistical thermodynamics, and the additivity of first and secondcoordination sphere mutational effects was verified. Spectroscopic studies (RR, NMR, UV-Vis, EPR) reveal that the electronic structure ofthe CuA site can be described by a double well potential energy surface related toalternative ground states. Moreover, relative populations of these states can bemodulated through mutations in the first and second coordination sphere, and throughpH in a particular point mutant. By means of protein film cyclic voltammetry, ET kineticparameters in both ground states could be independently established. The mutant M160H constitutes the first example of a model center in which two different groundstates can be selectively activated through a simple pH change. Finally, the characterization of type 1 (T1) sites artificially introduced in the Tt-CuAprotein backbone by loop engineering shows prominent differences with respect tonative T1 sites. This includes important spectroscopic changes and large redox potentialshifts; thus pointing out to the decisive role played by the protein matrix in definingfunctional (redox) and structural properties. These novel systems also show thecapability to bind exogenous ligands without losing its redox activity, allowing researchof this kind of phenomenon in a biological system.Fil: Zitare, Ulises Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Copper active sites in biology

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