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 heteronu clear 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 asy mmetric tensor is necessary to describe overall rotational diffusion of the protein, showing a significant improvement compared to analysis using eith er isotropic and axially symmetric tensors. This analysis indicates that th e 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 st ructure 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 pri ncipal axes with the symmetry axes of the dimeric structure derived from X- ray crystallography provides unambiguous evidence that the molecule is mono meric in the solution state. Lipari-Szabo-type mobility parameters extracte d when using the anisotropic description of overall tumbling are found to d iffer considerably from those found assuming isotropic global reorientation , where the internal dynamics of NH vectors present in helical regions of t he 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 dif ferent orientation of this helix compared to that found in the crystal stat e, possibly due to the volume of the different ligands present in the two f orms of the protein. Using restrained molecular dynamics and energy minimiz ation with respect to the relaxation rate ratios, we have quantified the di fference in the orientation of this helix and find that a significant reori entation is necessary to fulfill the measured relaxation rates.