Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin

Eukaryotic assimilatory nitrate reductase (NR) is a multi-domain protein that catalyzes the rate-limiting step in nitrate assimilation. This protein is highly conserved and has been extensively characterized in plants and algae. Here, we report hybrid NRs (NR2-2/2HbN) identified in two microalgal species, Heterosigma akashiwo and Chattonella subsalsa, with a 2/2 hemoglobin (2/2Hb) inserted into the hinge 2 region of a prototypical NR. 2/2Hbs are a class of single-domain heme proteins found in bacteria, ciliates, algae and plants. Sequence analysis indicates that the C-terminal FAD/NADH reductase domain of NR2-2/2HbN retains identity with eukaryotic NR, suggesting that the 2/2Hb domain was inserted interior to the existing NR domain architecture. Phylogenetic analysis supports the placement of the 2/2Hb domain of NR2-2/2HbN within group I (N-type) 2/2Hbs with high similarity to mycobacterial 2/2HbNs, known to convert nitric oxide to nitrate. Experimental data confirms that H. akashiwo is capable of metabolizing nitric oxide and shows that HaNR2-2/2HbN expression increases in response to nitric oxide addition. Here, we propose a mechanism for the dual function of NR2-2/2HbN in which nitrate reduction and nitric oxide dioxygenase reactions are cooperative, such that conversion of nitric oxide to nitrate is followed by reduction of nitrate for assimilation as cellular nitrogen.