Insights into Biomolecular Structure from Theoretical Spectroscopy

Abstract

The first part of the present PhD thesis is concerned with the reaction mechanism of Cytochrome P450 NO reductase. Two important aspects of this enzyme are studied. 1) The structural and electronic nature of the key intermediate I and 2) the unusual direct reduction of the active site by NADH.<br /> In the computational study the inclusion of the entire enzyme structure is important. For that purpose, an interface for quantum mechanics/molecular mechanics (QM/MM) calculations with the program packages ORCA and GROMACS is implemented. A QM/MM study is carried out using density functional theory (DFT) to describe the protein’s active site at quantum mechanical level (44–142 atoms) and a classical molecular mechanics force field to describe the remaining parts of the system (about 50000 atoms).<br /> Calculations on two experimentally well characterized intermediates of the reaction cycle are performed. Structural properties, Mössbauer parameters and IR calculations are in good agreement with the experimental data.<br /> The third intermediate of the reaction, the key intermediate I, can be observed only under limited NO concentration and is thus experimentally not well characterized. The structural and spectroscopic properties of six possible candidates for I are analyzed in detail. The calculated properties exclude four candidates, leaving two for additional analysis: the singlet diradicals Fe(III)-NHO•− and Fe(III)-NHOH•.<br /> In further QM/MM calculations two different reaction pathways, connecting all possible intermediates, are modeled. In order to perform these calculations the geometry optimization methodology in ORCA is extended by algorithms for constrained and transition state (TS) geometry optimization. Additionally, several approaches to exact Hessian matrices that are used in TS optimization are developed and implemented.<br /> The calculated kinetic and thermodynamic properties for both pathways are used for the kinetic simulation of the entire reaction course. The kinetic simulation helps to unravel the nature of intermediate I. It is identified as the singlet diradical species Fe(III)-NHOH•.<br /> For the QM/MM calculations of the reduction of heme-bound nitrosyl by the cofactor NADH the kinetic isotope effect as well as the calculated activation barrier are in agreement with the experimental data. The results reveal that the reduction is carried out in form of a hydride ion transfer. Additionally, they give insight into the factors that determine NADH binding in the active site and explain the loss of catalytic activity observed in site-directed mutagenesis experiments.<br /> The calculations on the formation of the N-N bond show that the mechanism behind the bond formation is spin-recoupling, which is only possible due to the diradical character of intermediate <em>I</em>.<br /> In a second part of the thesis a more general question is adressed. Namely, the validity of the point-dipole approximation, which is used in the EPR spectroscopy applications PELDOR (pulsed electron-electron double resonance) and DQC (double quantum coherence). Both techniques are used to assign distances in proteins and other macromolecules. The magnetic dipolar coupling between spin centers in macromolecules is measured experimentally in order to determine their distance and thus get important insight into the conformation of the entire molecule. For distance determination, a simple point-dipole approximation is frequently used. In the present thesis, DFT studies on a variety of model systems are performed to study the limitations of this approximation.<br /> The calculations show that delocalization of the spin density can lead to large errors in the obtained interspin distances. Even small delocalization leads to errors in the range of 1-2 Å. The deviations become dramatic for systems with large delocalization of the spin density, e.g. unsaturated linkers between the spin centers or aromatic nitroxide systems. For aromatic nitroxide systems there can even be a dependence on the orientation of the spin-carrying fragments.