Local stability of Rhodobacter capsulatus cytochrome c2 probed by solution phase hydrogen/deuterium exchange and mass spectrometry.
The hydrogen/deuterium exchange kinetics of Rhodobacter capsulatus cytochrome c2 have been determined using mass spectrometry. As expected, the relative domain stability was generally similar to that of the cytochrome c2 structural homolog, horse heart cytochrome c, but we were able to find evidence to support the presence of a second, small beta-sheet not found in the horse cytochrome, which stabilizes a structural region dominated by Omega loops. Importantly, we find that the so-called hinge region, comprised of 15 amino acids, which include the methionine sixth heme ligand (M96), is destabilized on oxidation, and this destabilization is propagated to a portion of the second Omega loop, most likely through perturbation of two hydrogen bonds that couple these two domains in the three dimensional structure. The mutation of a lysine at position 93 to proline amplifies the destabilization observed on oxidation of the wild-type cytochrome c2 and results in further destabilization observed in regions 52-60, 75-82, and 83-97. This suggests that hydrogen bond interactions involving two bound waters, the T94 hydroxyl, the front heme propionate and the Y75 hydroxyl, are significantly compromised upon mutation. In summary, these observations are consistent with the approximately 20-fold increase in the movement of the hinge away from the heme face in the oxidized cytochrome c2 as determined by ligand binding kinetics. Thus, H/D exchange kinetics can be used to identify relatively subtle structural features and at least in some cases facilitate the understanding of the structural basis of the dynamic properties of proteins.
Cheng
Guilong
G
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Wysocki
Vicki H
VH
Cusanovich
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D045362:Cytochromes c2 / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006736:Horses; D006859:Hydrogen / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization
D006859:Hydrogen; D045362:Cytochromes c2