Redox sensor properties of human cytoglobin allosterically regulate heme pocket reactivity

• Published in: Free radical biology & medicine Vol. 162; pp. 423 - 434
• Main Authors: DeMartino, Anthony W., Amdahl, Matthew B., Bocian, Kaitlin, Rose, Jason J., Tejero, Jesús, Gladwin, Mark T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021
• Subjects: Animals; Cysteine modification; Cytoglobin - metabolism; Functional midpoint potential; Globins - genetics; Heme - metabolism; Human cytoglobin; Humans; Hydrogen peroxide; Oxidation-Reduction; Protein Binding; Redox modulation; Stopped-flow kinetics; Human cytoglobin; Redox modulation; Functional midpoint potential; Hydrogen peroxide; Stopped-flow kinetics; Cysteine modification
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2020.10.321

Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of −189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding. [Display omitted] •Ubiquitously found mammalian hemoprotein cytoglobin is a redox sensor protein.•We measured cyanide binding kinetics to probe redox allostery of Cygb cysteines.•The midpoint potential of Cygb disulfide bond is in the biological redox range.•Mild oxidizing conditions trigger disulfide bond formation and enhance reactivity.