Rotational diffusion anisotropy and local backbone dynamics of carbon monoxide-bound Rhodobacter capsulatus cytochrome c′

The rotational diffusion and backbone dynamics of the carbon monoxide-bound Rhodobacter capsulatus cytochrome c' have been investigated using heteronuclear NMR spectroscopy. This protein consists of a four-helix bundle motif and a histidine-heme binding domain and has been shown to form a symmetric dimer in the crystal state. N-15 relaxation measurements reveal that an asymmetric tensor is necessary to describe overall rotational diffusion of the protein, showing a significant improvement compared to analysis using either isotropic and axially symmetric tensors. This analysis indicates that the molecule undergoes significant anisotropic reorientation with a diffusion tensor having principal components {1.37 +/- 0.05, 1.68 +/- 0.05, 2.13 +/- 0.07} x 10(7) s-(1). Hydrodynamic calculations performed on the crystal structure predict values of {1.400, 1.45, 2.12} x 10(7) s(-1) when a solvent shell of 3.0 Angstrom is included in the calculation. Comparison of the principal axes with the symmetry axes of the dimeric structure derived from X-ray crystallography provides unambiguous evidence that the molecule is monomeric in the solution state. Lipari-Szabo-type mobility parameters extracted when using the anisotropic description of overall tumbling are found to differ considerably from those found assuming isotropic global reorientation, where the internal dynamics of NH vectors present in helical regions of the molecule exhibit clear periodicity due to their orientation relative to the diffusion tensor. In addition, the relaxation properties of helix I are less well reproduced than those of the other three helices, implying a different orientation of this helix compared to that found in the crystal state, possibly due to the volume of the different ligands present in the two forms of the protein. Using restrained molecular dynamics and energy minimization with respect to the relaxation rate ratios, we have quantified the difference in the orientation of this helix and find that a significant reorientation is necessary to fulfill the measured relaxation rates.