Abstract
An important role of nitric oxide (NO) as either a free intermediate in the NH(3) oxidation pathway or a potential oxidant for NH(3) or NH(2)OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH(3) oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO(2)(-) production, whereas N. europaea grown under O(2) limitation produced NO equivalent to up to 40% of NO(2)(-) production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O(2) limitation abolished N(2)O production. These results indicate different turnover rates of NO during NH(3) oxidation under O(2)-replete and O(2)-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH(3) saturation and adaptation to O(2) limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N(2)O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O(2)-limited growth conditions.