A plant gene encoding one-heme and two-heme hemoglobins with extreme reactivities toward diatomic gases and nitrite

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.IV was a Formación de Personal Investigador fellow (BES-2015-073059) and EL is a Ramón y Cajal fellow (RYC2018-023867-I), both from the Spanish State Research Agency-Ministry of Economy, Industry and Competitiveness (MINECO). LM was the recipient of a fellowship partially funded by MIUR-Italy ('Progetto Dipartimenti di Eccellenza 2018-2022' allocated to Department of Chemistry 'Ugo Schiff'). This work was supported by grants AGL2017-85775-R and RTI2018-094623-B-C22 from MINECO, co-funded by the European Regional Development Fund, and by Government of Aragón (group A09_17R)