Nitrous oxide reduction in acidic environments

Popular science: In this project we have investigated the biogenesis of the nitrous oxide reductase (N2OR), the only enzyme that can neutralize potent greenhouse gas nitrous oxide (N2O). The N2O has a great global warming potential, much stronger than that of methane (CH4) and carbon dioxide (CO2). As we find ways to mitigate emissions of the two latter, the anthropogenic N2O release is rising and will continue to rise, unless actions are taken. The major source of anthropogenic N2O is agriculture, which directly links the emissions to food production as a result of intensive use of nitrogen fertilizers. The N2O contributes to ~ 35 % of the total climate forcing of food production. We have known for long that soil N2O emissions are affected by several factors, and the pH of the soil is one of the key controllers, meaning that the lower the soil pH, the more N2O is liberated to the atmosphere. Our aim in this project was to reveal how pH affect the synthesis of the N2OR protein for better understanding of the problem and for finding mitigations options for N2O emissions. We have used an interdisciplinary approach joining forces of two strong laboratories in Europe. Collaborative work between NMBU and University of Freiburg delivered results that explain why the N2OR is less active in acidic soils. The enzyme does not mature properly at pH < 6, it lacks copper ions which are critical for its function. Studies of model organisms showed that there is no shortage of copper in cells grown at low pH, but the N2OR does not get it. Our findings stress the importance of soil pH management as a standard routine, not only for better productivity but also for lowering the N2O greenhouse gas emissions.

The overall outcome of the project has been very successful. Despite some hurdles due to pandemics and initial difficulties, the objectives have been accomplished. I have gained a lot of experience and new expertise during my stay in Freiburg. Both partners UNI-FR and NMBU have gained from this collaborative work, as seen from join publications and plans for future collaborative work.There has been extensive transfer of knowledge both ways, our group in Ås, in particular, is now capable of conducting more refined biochemical studies investigating proteins, which have already been implemented in the work of two PhD candidates that I am co-supervising. The scientific outcome of the project. We have revealed the details of N2OR biogenesis under sub-optimal low pH conditions, corroborating our hypothesis that low pH hinders the Cu maturation of the protein. This solid and convincing results will help to understand the importance of soil pH management as a measure to mitigate agriculture-related N2O greenhouse gas emissions.

Nitrous oxide (N2O) is a potent greenhouse gas and the dominant ozone-depleting substance emitted in the XXI century. Several microbially mediated processes within the nitrogen cycle can generate N2O. Of these, denitrification is the dominant source in most ecosystems, and incomplete soil denitrification, in which nitrate or nitrite is reduced only to N2O, accounts for up to 60 per cent of the global emissions of this gas. Soil pH is one of the major controllers of N2O emissions, seen from the strong, negative correlation between soil acidity and N2O/ (N2O+N2) product ratio. The only known biological sink for N2O is the multi-copper enzyme nitrous oxide reductase (NOS) that reduces it to harmless N2. Assembly and copper maturation of NOS take place in the periplasmic space, requiring a complex machinery of accessory factors. Studies of the N2O emissions using the model organism Paracoccus denitrificans reproduced the effect of low pH observed in fields, and identified a plausible explanation: low pH impedes the synthesis of functional NOS, rather than its function, since NOS synthesized at high pH functioned well at low pH. Our working hypothesis to explain the lack of N2O reduction in bacteria growing in acidic pH conditions, is that the maturation of the NOS apoprotein is affected by H+ ions after its transportation to the periplasm. Recently we proved that NOS is not only synthesized at acidic pH, but also transported to the periplasm, which effectively pins down the pH-effect to impede maturation of NOS in the periplasm, as originally hypothesized. In this interdisciplinary project the ecophysiology and structural biology will be combined to unravel mechanisms causing N2O emissions from acidic environment. We expect that the investigation of the hitherto unknown efficient N2O reduction in Rhodanobacter will reveal substantial differences in NOS itself and/ or its maturation process that will provide clues for possible mitigation options for N2O greenhouse gas.