Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite

Abstract

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins.

Keywords: hemoglobin; nitric oxide; nitrite; nitrite oxidase activity; nitrite reductase activity; nonsymbiotic hemoglobin; phytoglobins; redox reactivity.

Figure 1

The two representative folding structures for nonsymbiotic phytoglobins from Arabidopsis thaliana (AtHb1, 3ZHW, pdb code) and the truncated one from the same organism (AtHb3, 4C0N, pdb code). The coordination differences are strikingly visible. While AtHb1 is hexacoordinated and has a 3/3 fold (helices A, E, F on B, G, H), AtHb3 is pentacoordinate and has a 2/2 fold (helices B, E on G, H). Figure reproduced here with permission from [48].

Figure 2

UV-vis spectral changes for the titration of recombinant AtHb3 methemoglobin (HbFe³⁺) with nitrite in aerobic conditions. UV-vis spectra of 8 μM methemoglobin (HbFe³⁺) after the addition of nitrite at different concentrations from 5 μM to 30 mM in 50 mM phosphate buffer at pH 7 and 25 °C. (A) Spectral monitoring for the full 280–780 nm domain. The vertical black arrows indicate the directions of the spectral features that exhibit striking changes, and the upward red arrow at 360 nm indicates the nitrite band that increases in absorbance as its concentration rises. The 480–780 nm domain was multiplied with a factor of 5 for better visibility. The initial spectrum (without nitrite) is colored in red, and the final spectrum (at 30 mM nitrite) is colored in blue, whereas at intermediate nitrite concentrations, the spectra are colored in hues of green, from light green (low concentration of nitrite) to darker green (high concentration of nitrite). (B) Titration curve at 417 nm (specific for AtHb3Fe³⁺-NO₂⁻ adduct) and the value for K_D as determined by sigmoidal fitting for log concentration axis.

Figure 3

The reaction between nitrite and recombinant Arabidopsis thaliana nonsymbiotic deoxyhemoglobins (HbFe²⁺) and horse heart deoxyMb. UV-vis spectra of 8 μM deoxyhemoglobins (HbFe²⁺) during oxidation by nitrite in 50 mM phosphate buffer at pH 7 and 25 °C; (A) 8 μM deoxyAtHb1 with 0.05 mM nitrite; (B) 8 μM deoxyAtHb2 with 0.1 mM nitrite; (C) 8 μM deoxyAtHb3 with 0.25 mM nitrite; and (D) 8 μM deoxyMb with 0.5 mM nitrite. In all cases, 5 mM dithionite was used to ensure an anaerobic medium and the reduction of the formed ferric forms. The specific monochromatic color for each form of Hb is transient from a light hue to a dark hue. The 480–780 nm domain was multiplied with a factor of 5 for better visibility. The vertical black arrows indicate the directions of the spectral features that exhibit striking changes. Insets: the kinetic profile of the reactant consumption (blue) and the kinetic profile of the product formation (red) are shown.

Figure 4

Kinetics of nitrite reaction with Arabidopsis thaliana nonsymbiotic deoxyhemoglobins (HbFe²⁺) and horse heart deoxyMb. (A) Time course of the reaction of each Hb and Mb at 1 mM nitrite. The absorbance change is normalized to that associated with the transition from each deoxyhemoglobin (413 nm for AtHb1, 412 nm for AtHb2, 419 nm for AtHb3, and 422 for Mb) to its endpoint spectrum. (B) Plot of the observed rate constants (k_obs/min⁻¹) versus nitrite concentration obtained in 50 mM phosphate buffer at pH 7 and 25 °C.

Figure 5

Kinetic profiles and their corresponding lag times and maximum decay rates for the reaction between oxyAtHb1 and nitrite. (A) At different concentrations of sodium nitrite, 8 μM oxyAtHb1 was incubated with NaNO₂ (1.47–2.12 mM) in 50 mM phosphate buffer (pH 7). (B) At different pH values, 8 μM oxyAtHb1 was incubated with 1.83 mM NaNO₂ in a buffer with pH 6.8–7.3.

Figure 6

EPR spectra of the studied met form of the Hbs (200 μM) before and after cca. 1 min of NaNO₂ treatment. The spectra were measured in 50 mM phosphate buffer (pH 7, temperature 100 K, X-band), and the cavity spectrum was subtracted from all spectra. For comparison, in the case of AtHb3, the deoxy (in blue) and oxy (in green) forms were also included. The spectrum of the deoxy form after nitrite treatment was divided by 2 so that it better fits the scale.

Figure 7

EPR spectra of the deoxyHbs (200 μM) after cca. 1 min of NaNO₂ treatment in 50 mM phosphate buffer (pH 7, temperature 100 K, X-band). The g values and coupling constants are indicated. Note that Mb shows no detectable features, and the expected ferrous Mb nitrosyl is presented as a reference (green). Black dots in the top spectrum have no other meaning except for the identification of the gridlines added for the x-axis.

Figure 8

Tentative mechanism illustrating both nitrite reductase activity for deoxyHbs and nitrite oxidase activity for oxyHbs. The dashed arrows indicate the more complex reactions that take place in several steps, with potential reaction intermediates not being depicted here.