THB1, a truncated hemoglobin, modulates nitric oxide levels and nitrate reductase activity

Abstract

These authors contributed equally to this work. SUMMARYHemoglobins are ubiquitous proteins that sense, store and transport oxygen, but the physiological processes in which they are implicated is currently expanding. Recent examples of previously unknown hemoglobin functions, which include scavenging of the signaling molecule nitric oxide (NO), illustrate how the implication of hemoglobins in different cell signaling processes is only starting to be unraveled. The extent and diversity of the hemoglobin protein family suggest that hemoglobins have diverged and have potentially evolved specialized functions in certain organisms. A unique model organism to study this functional diversity at the cellular level is the green alga Chlamydomonas reinhardtii because, among other reasons, it contains an unusually high number of a particular type of hemoglobins known as truncated hemoglobins (THB1-THB12). Here, we reveal a cell signaling function for a truncated hemoglobin of Chlamydomonas that affects the nitrogen assimilation pathway by simultaneously modulating NO levels and nitrate reductase (NR) activity. First, we found that THB1 and THB2 expression is modulated by the nitrogen source and depends on NIT2, a transcription factor required for nitrate assimilation genes expression. Furthermore, THB1 is highly expressed in the presence of NO and is able to convert NO into nitrate in vitro. Finally, THB1 is maintained on its active and reduced form by NR, and in vivo lower expression of THB1 results in increased NR activity. Thus, THB1 plays a dual role in NO detoxification and in the modulation of NR activity. This mechanism can partly explain how NO inhibits NR post-translationally.

Nitrogen-induced metabolic changes and molecular determinants of carbon allocation in Dunaliella tertiolecta

“…The top 10 most highly expressed or repressed genes were related to nitrogen-scavenging or photosynthesis (Table S3). For instance, THB1, a truncated hemoglobin highly expressed in the presence of nitric oxide37, was repressed by almost 300-fold. Likewise, three subunits of the urea active transporter were upregulated between 52- and 72-fold, representing one of the many enzymes whose expression are known to increase to harvest nitrogen from nitrogen-containing compounds such as ammonium, nitrate, nitrite, urea, purines, pyrimidines and amino acids38.…”

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite‐dependent NO production in Chlamydomonas

“…As shown herein, both are needed to synthesize NO from nitrite in vivo , so that the deficient functionality of either NR or ARC would lead to the derepression of the system, which tries to compensate the decreased NO levels. It has been previously shown that high NO leads in Chlamydomonas to repression of nitrate assimilation related genes ( NIA1 , NRT2.1, or THB2 ) (de Montaigu et al ; Sanz‐Luque et al ). Thus ARC expression seems to be also negatively regulated by NO, because the lack of NO production causes ARC derepression (Fig.…”

“…), and second, promoting reduction of THB1 for NO conversion to nitrate. THB1 is a truncated haemoglobin, which has NO‐dioxygenase activity, when reduced, and NR seems to mediate also this reduction (Sanz‐Luque et al ). In this way, by distributing electrons to ARC and THB1, NR would be playing a central role in the control of NO amounts in the cell for signalling of numerous processes.…”

Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1

“…However with the current data, we cannot discriminate which ones of these alternatives would be the correct one. Interestingly, crARC seems to have basic amino acids in the entire outer part where the Mo Cofactor is bound, and it has been described that there are mostly acid amino acids in the outside of crCitb5-1 and crCitb5-R [35], so this would explain in part the interaction between these three proteins.…”