Accurate Structure andDynamics of the Metal-Siteof Paramagnetic Metalloproteins from NMR Parameters Using NaturalBond Orbitals

摘要

A natural bond orbital (NBO) analysis of unpaired electron spin density in metalloproteins is presented, which allows a fast and robust calculation of paramagnetic NMR parameters. Approximately 90% of the unpaired electron spin density occupies metal–ligand NBOs, allowing the majority of the density to be modeled by only a few NBOs that reflect the chemical bonding environment. We show that the paramagnetic relaxation rate of protons can be calculated accurately using only the metal–ligand NBOs and that these rates are in good agreement with corresponding rates measured experimentally. This holds, in particular, for protons of ligand residues where the point-dipole approximation breaks down. To describe the paramagnetic relaxation of heavy nuclei, also the electron spin density in the local orbitals must be taken into account. Geometric distance restraints for ¹⁵N can be derived from the paramagnetic relaxation enhancement and the Fermi contact shift when local NBOs are included in the analysis. Thus, the NBO approach allows us to include experimental paramagnetic NMRparameters of ¹⁵N nuclei as restraints in a structure optimizationprotocol. We performed a molecular dynamics simulation and structuredetermination of oxidized rubredoxin using the experimentally obtainedparamagnetic NMR parameters of ¹⁵N. The corresponding structuresobtained are in good agreement with the crystal structure of rubredoxin.Thus, the NBO approach allows an accurate description of the geometricstructure and the dynamics of metalloproteins, when NMR parametersare available of nuclei in the immediate vicinity of the metal-site.