Disulfide bonds regulate binding of exogenous ligand to human cytoglobin

Abstract

Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.

Peroxidase activation of cytoglobin by anionic phospholipids: Mechanisms and consequences

“…To determine the oxidation state of the thiol groups and the presence or absence of the disulfide bond between Cys38 and Cys83 we ran the Cygb samples untreated or after incubation with increasing amounts of DTT. As shown in Supplementary Figure 2, the Cygb monomer forms two discernible bands that we ascribe to a difference in the thiol reduction state, in agreement with previous reports [11, 12]. The upper band corresponds to the protein with the reduced thiols, whereas the protein with the intramolecular disulfide bond formed gives a band at a lower molecular weight.…”

“…This effect of anionic lipids is realized via triggering structural re-arrangements of the protein leading to its transition from the hexa-coordinated to penta-coordinated heme-iron state. Since the discovery of Cygb in 2001, several studies have documented the importance of the thiol to disulfide conversions of Cys38/Cys83 in controlling heme coordination and the binding of diatomic ligands [9, 12, 30] as well as lipids [10, 11], thus emphasizing possible redox mechanisms of its regulation. Recently, the peroxidase activity of Cygb has been described [31], albeit at supra-physiological concentrations of H 2 O 2 [31].…”

“…However, the hexa- and penta-coordinated forms are present in a dynamic equilibrium, and both proteins can stabilize their penta-coordinated states upon oxidation of their Cys residues to an internal disulfide [9-12]. Recent work has established that, similarly to Cyt c , Cygb can also stabilize the penta-coordinate species by lipid binding, and thus hypothetically promote its peroxidase activity [13].…”

Coupling of disulfide bond and distal histidine dissociation in human ferrous cytoglobin regulates ligand binding

“…The affinities of the monomer S-S and monomer S-H are high (K D < 10 À7 M) and could not be accurately determined from this method. The affinity of the dimer S-S is, however, significantly lower and we estimate from the fit a K D of 3.3 ± 0.9 Â 10 À7 M. These results are consistent with those reported by Tsujino et al [10].…”

“…Through timeresolved spectra we have assigned unequivocally CO binding to hexa-and penta-coordinate forms and have made direct measurement of histidine rebinding following photolysis. We present a model that summarizes our conclusions and those of earlier work, which incorporates changes in ligand affinities, ligand binding rate constants and distal histidine association and dissociation rate constants controlled by the cysteine residue redox state in the monomeric form [9,10,12].…”