The Redox State of the Cell Regulates the Ligand Binding Affinity of Human Neuroglobin and Cytoglobin

Abstract

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.

Slow Ligand Binding Kinetics Dominate Ferrous Hexacoordinate Hemoglobin Reactivities and Reveal Differences between Plants and Other Species

“…It has also been shown that the disulfide bond in the CD region can affect the kinetics of hexacoordination by lowering k -H for Ngb (9). Our results suggest that this effect is not the same for Cgb, where an increase in k -H was observed in the presence of DTT.…”

“…After a treatment of 24 h at 4°C with 10 mM DTT (as described by Hamdane et al (9)), the protein solutions were reacted with 1 mM CO. The resulting time course for Ngb is indeed slower but still exhibits two different phases of binding with nearly equal amplitudes.…”

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…3A). These forms might represent atypical migrations of stable protein conformations and it has been hypothesized that rupture/ formation of the intra molecular (CD7-B5) disulfide bond of NGB [50] can give rise to different apparent molecular mass forms [49]. Posttranslational modifications could also occur, such as covalent links of cofactors or phosphorylation.…”

Functional Properties of Neuroglobin and Cytoglobin. Insights into the Ancestral Physiological Roles of Globins

“…These results argue against a role for mammalian Ngbs as an intracellular O 2 store. The lower P 50 value that was originally recorded for human Ngb was presumably due to in vitro formation of an internal disulfide bridge in the protein (20), which increases the affinity for O 2 (see below). We have also shown that, in contrast to Mb, the O 2 affinity of Ngb is markedly sensitive to changes in pH, and that, depending on the temperature range, both normal and reverse Bohr effects are present (indicating an increase or a decrease in the O 2 affinity, respectively, with a pH increase) (19).…”

“…A common feature of vertebrate Ngbs is the presence of two to three solvent-accessible reactive cysteine residues (19,20). These are located at the level of the CD corner and D helix (one or two cysteines) or in the G helix (one cysteine) of the protein.…”