Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.
All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.
Dumortier
C
C
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Fitch
J
J
Van Petegem
F
F
Vermeulen
W
W
Meyer
T E
TE
Van Beeumen
J J
JJ
Cusanovich
M A
MA
D045362:Cytochromes c2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008715:Methionine / Q000378:metabolism*; D009154:Mutation; D016354:Rhodobacter capsulatus / Q000201:enzymology*
D007093:Imidazoles; D008024:Ligands; D045362:Cytochromes c2; D008715:Methionine