Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes

Abstract

Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O2 and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. The reaction of reduced deoxyNgb with O2 and NO is slow (t(1/2) approximately 2 s) and ligand concentration-independent, because exogenous ligand binding can only occur upon dissociation of the distal His-64, which is coordinated to the ferrous heme iron. By contrast, NgbO2 reacts very rapidly with NO, yielding metNgb and NO3- by means of a heme-bound peroxynitrite intermediate. Steady-state amperometric experiments show that Ngb is devoid of O2 reductase and NO reductase activities. To achieve this result, we have set up a protocol for efficient reduction of metNgb using a mixture of FMN and NADH under bright illumination. The results are discussed with reference to a global scheme inspired by the 3D structures of metNgb and NgbCO. Based on the ligand-linked conformational changes discovered by crystallography, the pathways of the reactions with O2 and NO provide a framework that may account for the involvement of Ngb in controlling the activation of a protective signaling mechanism.

Fig. 1.

Reactions with O₂ and NO. (A) Overall scheme of some reactions of Ngb. The species on the left (reduced) and those on the right (met or ferric) are hexacoordinated by His-96 and His-64 (highlighted in blue); the other three species (highlighted in red) are supposed to have the 3D structure determined for NgbCO (17), lacking the internal coordination bond with His-64. The reduced pentacoordinate form is a transient species not significantly populated at equilibrium. (B) Overall time course of the reaction of reduced Ngb with O₂ (red) and NO (black) at pH 7.0 and 20°C. The reaction is slightly heterogeneous, the overall rate coefficient being k′ ≈0.4 s^-1; it may be appreciated that the rate is NO concentration-independent. (C) View of the active site of murine Ngb in the met (blue) and CO-bound (red) state. Upon CO ligation, the coordination bond with His-64 (above the heme plane) is broken. The imidazole ring moves only slightly, whereas the heme tilts and slides toward a preexisting space (17). This shift is associated with (i) a substantial change of the position of the proximal His-96 (seen at the bottom) and (ii) the disappearance of the proximal branch of the large cavity (contour highlighted in blue) and its extension on the distal side (indicated with orange contour toward the left).

Fig. 2.

The reaction of ferrous oxygenated Ngb with NO. deoxyNgb was obtained by incubating degassed metNgb for a few minutes with 400 μM NADH plus 1 μM FMN under bright white light illumination. Anaerobic conditions were ensured by addition of 2 mM glucose, 8 units/ml glucose oxidase, and 260 units/ml catalase. In the stopped-flow apparatus operating in the sequential mixing mode, deoxyNgb was first mixed with air-equilibrated buffer (50 mM phosphate buffer/20 μM EDTA, pH = 7) to obtain NgbO₂ after a suitable delay time (30 s or 100 s at 20°C or 5°C, respectively); thereafter, this solution was mixed with an anaerobic solution containing NO. NgbO₂ reacted with NO after the second mixing, yielding in the dead-time an intermediate (spectroscopically different from NgbO₂) that decays to metNgb. (A) Time course of decay of the intermediate to metNgb at 5°C and 20°C, as obtained from the absorption changes at 399–419 nm after normalization. Fitted rate constants: k ≈ 300 s^-1 at 20°C and [NO] = 10 μM(▪) or 250 μM(□); k ≈ 85–95 s^-1 at 5°C and [NO] = 10 μM (○), 40 μM (•), or 200 μM (▵). Experiments were carried out at [Ngb] ≈ 4.5 μM or [Ngb] ≈ 16 μM (final concentration). (Inset) Difference absorption spectra collected at 20°C over the first 25 ms, with reference to the spectrum of metNgb. [NO] = 250 μM (final concentration). The arrows indicate the progress of the reaction. (B and C) Absolute absorption spectra in the Soret (B) and in the visible region (C). The thick line represents the intermediate formed in the dead-time of the stopped flow (2.5 ms) calculated at t = 0. The thin line represents the endpoint species of the decay (see A), corresponding to metNgb. The dotted line represents NgbO₂. The peak wavelengths, λ_max, for the three species are indicated in B.

Scheme 1.

Fig. 3.

Ngb reduction by photoactivated NADH/FMN. (A) Reduction of metNgb by NADH alone. Shown are absorption spectra collected from 2.5 ms to 50 s after mixing metNgb (≈8 μM) with NADH (2 mM) in the diode array stopped-flow apparatus. (Inset) Best fit of the reaction time course as obtained by singular value decomposition analysis; k ≈ 0.1 s^-1 for the main kinetic component. (B) Dependence of the rate coefficient for Ngb reduction on the concentration of FMN in the presence of 1 mM NADH and under illumination by white light. Conditions were as described for Fig. 2 (t = 20°C).

Fig. 4.

Testing the O₂ reductase and NO reductase activity of Ngb. Measurements were carried out at room temperature either in the dark or under bright white light illumination of the reaction chamber to ensure an efficient, steady reduction of Ngb by the NADH/FMN mixture. (A) O₂ consumption measurement under illumination. After addition of NADH and FMN, the addition of several aliquots of metNgb does not significantly affect the O₂ consumption rate. Fast response of the electrode is demonstrated by addition of dithionite. In comparison, addition of 0.2 μM beef heart cytochrome c oxidase in the presence of excess reductant (ascorbate, tetramethyl-p-phenylene diamine, and cytochrome c) would cause O₂ exhaustion in <30 s under similar experimental conditions. (B) NO measurement. After addition of three aliquots of NO (final concentration ≈ 30 μM) to degassed buffer, NO consumption was monitored either in the dark or under illumination. The addition of degassed NADH and FMN accelerates NO consumption in both cases, with a larger effect under illumination; however, the subsequent addition of anaerobic metNgb has essentially no effect on the rate of NO consumption. In the dark, residual NO in solution is promptly scavenged by addition of dithionite. In comparison, addition of 2 μM bacterial NO reductase in the presence of excess ascorbate and tetramethyl-p-phenylene diamine would cause NO to be completely consumed in ≈1 s under similar experimental conditions.

Scheme 2.