Characterization of the Function of Cytoglobin as an Oxygen-Dependent Regulator of Nitric Oxide Concentration

Abstract

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an O(2)-dependent manner. This regulates NO levels in the vascular wall; however, the underlying molecular basis of this O(2)-dependent NO consumption remains unclear. While cytoglobin (Cygb) was discovered a decade ago, its physiological function remains uncertain. Cygb is expressed in the vascular wall and can consume NO in an O(2)-dependent manner. Therefore, we characterize the process of the O(2)-dependent consumption of NO by Cygb in the presence of the cellular reductants and reducing systems ascorbate (Asc) and cytochrome P(450) reductase (CPR), measure rate constants of Cygb reduction by Asc and CPR, and propose a reaction mechanism and derive a related kinetic model for this O(2)-dependent NO consumption involving Cygb(Fe(3+)) as the main intermediate reduced back to ferrous Cygb by cellular reductants. This kinetic model expresses the relationship between the rate of O(2)-dependent consumption of NO by Cygb and rate constants of the molecular reactions involved. The predicted rate of O(2)-dependent consumption of NO by Cygb is consistent with experimental results supporting the validity of the kinetic model. Simulations based on this kinetic model suggest that the high efficiency of Cygb in regulating the NO consumption rate is due to the rapid reduction of Cygb by cellular reductants, which greatly increases the rate of consumption of NO at higher O(2) concentrations, and binding of NO to Cygb, which reduces the rate of consumption of NO at lower O(2) concentrations. Thus, the coexistence of Cygb with efficient reductants in tissues allows Cygb to function as an O(2)-dependent regulator of NO decay.

Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions

“…The observed k on /k off is 165 at 37°C (pH 7.0) (48), and thus ϳ99.4% of Cygb is present as a six-coordinate that cannot react and scavenge NO. Compared with Mb and Hb, Cygb is a much weaker NO scavenger with only 0.6% of Cygb present as a fivecoordinate to react with and trap NO as shown in Reaction 5, Fe 2ϩ Ϫ Cygb Ϫ His ϩ NO -| 0…”

Effect of temperature, pH and heme ligands on the reduction of Cygb(Fe3+) by ascorbate

“…The expression and purification process of Cygb was described in our prior reports [24, 25]. The reduction of Cygb(Fe 3+ ) by Asc was performed under anaerobic conditions and monitored using a Cary 50 UV/Vis spectrophotometer by scanning from 350 nm to 650 nm or by fixing the wavelength at the absorbance peak of the corresponding absorption bands to measure the changes in absorbance with time.…”

“…This recycling process for Cygb can allow Cygb to efficiently metabolize NO at low micromolar Cygb concentrations. The rate of consumption of NO by Cygb is O 2 -dependent [21, 24] and limited by reducing Cygb(Fe 3+ ) to Cygb(Fe 2+ ) [21]. …”

“…We have recently proposed a reaction mechanism to describe how Cygb regulates the rate of NO consumption in an O 2 -dependent manner and suggested that Cygb may act as an oxygen sensor in the vascular wall to regulate the NO decay rate and diffusion distance in response to changes in O 2 concentration [24]. We can see from this reaction mechanism that the rate of NO metabolism by Cygb is largely affected by the rate of Cygb reduction by Asc, the key control step in the process of Cygb-mediated NO metabolism.…”

Redox and Peroxidase Activities of the Hemoglobin Superfamily: Relevance to Health and Disease

“…Cygb has been proposed to act as an oxygen diffusion proteins similar to Mb, based on observations of increased expression of Cygb in heart and muscle in amphibians that lack Mb (126). Cygb has also been proposed to function as an NO scavenger, from observed high rates of NO consumption and its fast re-reduction by ascorbate and cytochrome P450 reductase (64). This proposal of NO consumption has also been enhanced by increased NO levels and decreased intracellular nitrate production from partially silenced Cygb (41).…”