Ammonia oxidation in autotrophic nitrification is a complex process that requires several enzymes as well as oxygen as a reactant. The key enzymes necessary for releasing energy during oxidation of ammonia to nitrite are ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). The first is a transmembrane copper protein which catalyzes the oxidation of ammonia to hydroxylamine () taking two electrons directly from the quinone pool. This reaction requires O.
The second step of this process has recently fallen into question. For the past few decades, the common view was that a trimeric multiheme c-type HAO converts hydroxylamine into nitrite in the periplasm with production of four electrons (). The stream of four electrons is channeled through cytochrome c to a membrane-bound cytochrome c. Two of the electrons are routed back to AMO, where they are used for the oxidation of ammonia (quinol pool). The remaining two electrons are used to generate a proton motive force and reduce NAD(P) through reverse electron transport.
Recent results, however, show that HAO does not produce nitrite as a direct product of catalysis. This enzyme instead produces nitric oxide and three electrons. Nitric oxide can then be oxidized by other enzymes (or oxygen) to nitrite. In this paradigm, the electron balance for overall metabolism needs to be reconsidered.