Nitrous Oxide Emissions from Nitrite Are Highly Dependent on Nitrate Reductase in the Microalga Chlamydomonas reinhardtii

Nitrous oxide (N(2)O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N(2)O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N(2)O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N(2)O and CO(2) in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N(2)O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N(2)O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N(2)O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO(2) emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO(2) fixation and release.