Cytoglobin conformations and disulfide bond formation

Abstract

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…4) shows a pattern similar to the one observed for Ngb [146,162]. Photodissociated ligands can exit to the solvent following separate pathways, some of which exploit temporary docking sites [162] Hexacoordination by the distal His residue imposes large changes in energetic barriers that were suggested to modulate the flux of reactants to and products from the reaction site at the heme.…”

“…Gel encapsulation and bulk viscosity have profound effects on the exchange of ligands between the protein matrix and the solvent. Good examples to illustrate this concept are provided by non symbiotic plant hemoglobins (nsHb) [141,142], neuroglobin (Ngb) [143,144] and cytoglobin (Cygb) [145,146]. These Hbs are collectively termed hexacoordinate Hbs because in the absence of hexogenous diatomic ligands, Fe coordination is completed by the distal His residue [147].…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…The structure of human Cygb shows that the dimer is stabilized by two inter-protein disulfide bridges between Cys 38 and Cys 83 and through interactions between the center of the E-helix and the AB corner (Sugimoto et al, 2004), whereas the dimer interface is formed by hydrophobic contacts between Phe 53 and Ile 126 in the crystal structure of a Cygb mutant with both Cys residues replaced by Ser . The presence of the inter-protein disulfide bridge in Cygb is not supported by solution studies (Hamdane et al, 2003;Lechauve et al, 2010). Lechauve et al (2010) have shown that in the absence of a reducing agent, Cys 38 and Cys 83 form an intra-protein disulfide bridge connecting helices B and E. The intra-protein disulfide bond reduces the affinity of the distal histidine for the heme iron by approximately two-fold and consequently alters the affinity for O 2 binding (Hamdane et al, 2003).…”

“…The presence of the inter-protein disulfide bridge in Cygb is not supported by solution studies (Hamdane et al, 2003;Lechauve et al, 2010). Lechauve et al (2010) have shown that in the absence of a reducing agent, Cys 38 and Cys 83 form an intra-protein disulfide bridge connecting helices B and E. The intra-protein disulfide bond reduces the affinity of the distal histidine for the heme iron by approximately two-fold and consequently alters the affinity for O 2 binding (Hamdane et al, 2003). Comparable modulation of the equilibrium constant for distal histidine was also observed in Ngb, even though the Cys residues involved in the formation of the disulfide bond are located in the flexible CD loop and a short D helix (Dewilde et al, 2001;Hamdane et al, 2003).…”

“…The disruption of the disulfide bond by the mutation of Cys38 and Cys83 by Ser may result in different subunit orientations from the ones observed in the native protein (Makino et al, 2006). Noteworthy, it was proposed that only a small fraction of dimer might be formed in near-physiological conditions in vitro (Lechauve et al, 2010).…”