Plant hemoglobins can be maintained in functional form by reduced flavins in the nuclei and confer differential tolerance to nitro-oxidative stress

39 Pags., 1 Tabl., 5 Figs., with Supplementary Supporting Information (1 Suppl. Tabl., 5 Suppl. Figs.). The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-313XThe heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O2 and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and ‘truncated’ (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O2 affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O2 scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.This work was funded by MINECO-FEDER (grant AGL2011-24524) and Gobierno de Aragón-FSE (group A53). M.S. was supported by a predoctoral contract from Junta de Ampliación de Estudios-CSIC.Peer reviewe