Effects of extrinsic imidazole ligation on the molecular and electronic structure of cytochrome c

Abstract

Although imidazole ligand binding to cytochrome c is not directly related to its physiological function, it has the potential to provide valuable information on the molecular and electronic structure of the protein. The solution structure of the imidazole adduct of oxidized horse heart cytochrome c (Im-cyt c) has been determined through 2D NMR spectroscopy. The Im-cyt c, 8 mM in 1.2 M imidazole solution at pH 5.7 and 313 K, provided altogether 2,542 NOEs (1,901 meaningful NOEs) and 194 pseudocontact shifts. The 35 conformers of the family show the RMSD values to the average structure of 0.063+/-0.007 nm for the backbone and 0.107+/-0.007 nm for all heavy atoms, respectively. The characterization of Im-cyt c is discussed in detail both in terms of structure and electronic properties. The replacement of the axial ligand Met80 with the exogenous imidazole ligand induces significant conformation changes in both backbone and side chains of the residues located in the distal axial ligand regions. The imidazole ligand binds essentially parallel to the imidazole of the proximal histidine, the two planes forming an angle of 8+/-7 degrees. The electron delocalization on the heme moiety and the magnetic susceptibility tensor are consistent with these structural features.

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides

“…Ligand Binding to c-type Hemes-The binding of small molecules to c-type hemes has been widely studied in type I cytochromes c, where displacement of the native methionine ligand is accompanied by conformational changes in the heme binding domain (13,14,45). The mechanism of binding is still under debate, but the equilibrium properties are well established for many small molecules and depend on the nature of the exogenous ligand and the environment provided by the heme pocket.…”

“…In most cases, binding occurs preferentially to the ferric iron and causes substantial changes in the redox potential (8,9) and spectral properties of the cytochrome (10 -12). These include cyanide, azide, fluoride, imidazole, pyridine, and nitric oxide (8,9,(13)(14)(15). For example, binding of imidazole (Im) to oxidized mitochondrial cyt c or bacterial cyt c 2 produces a blue shift in the Soret region as well as changes in a number of weak absorbance bands between 450 and 600 nm (10 -12).…”

“…Atom coordinates for simulation of horse heart cyt c with Im were taken from the Protein Data Bank 1FI7 NMR structure (14). For cyt c 2 , the coordinates were from the Protein Data Bank 1CXA x-ray structure (13).…”

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides

“…The binding of small molecules to c-type hemes has been widely studied in type I cytochromes c and is known to result in displacement of the native methionine ligand and to cause significant conformational changes in the heme binding domain (1)(2)(3)(4). The affinities and kinetics of binding reflect the interactions among the exogenous ligand, the heme, and the protein and provide a window into the static and dynamic properties of the heme domain.…”

“…Structural studies of cyts c and c 2 with exogenous ligands bound (1,2,5) show that part of the linker sequence (termed the "hinge" by Dumortier et al (3)) is rotated to create a slight opening or loosening of the heme binding cavity. The initial and final states are generally referred to as closed and open.…”