Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide

Abstract

Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (<400 ,uM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazineoxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent 02 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.The major process in microbial nitrification is the oxidation of ammonia to nitrite, followed by the oxidation of nitrite to nitrate. These reactions are predominantly catalyzed by specialized autotrophic bacteria characterized by such species as the ammonia-oxidizing bacterium Nitrosomonas europaea and the nitrite-oxidizing bacterium Nitrobacter winogradskyi, respectively. Although our understanding of the biochemistry and enzymology of individual reactions in the nitrification process is far from complete, there is a considerable general interest in nitrification because it plays an important role in controlling the balance between fixed and mobile forms of nitrogen in soil and water systems. For example, in agriculture, nitrification can lead to substantial losses of costly ammonia-based fertilizer (and urea-based fertilizer, which hydrolyzes to ammonia) because both nitrification products, nitrite and nitrate, are easily leached from soils by water. In addition, nitrate is the major substrate for denitrifying bacteria that generate gaseous products (N20, NO, and N2) which are lost to the atmosphere. These same reactions, while detrimental to agriculture, are regarded as positive benefits in water treatment regimens, in which the objective is to reduce the nitrogen content of waste waters (22).Because it is ammonia-oxidizing bacteria that initiate nitrification as a whole, there has been considerable research into the inhibition of this process. Ammonia oxidation by N. europaea is a two-stage process that is now thought to involve the initial oxidation of ammonia to hydroxylamine by a membrane-bound enzyme known as ammonia monooxyge...

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper

“…The time-dependent inactivation of AMO by ['4Clacetylene results in a progressive and saturable incorporation of 14C label into the 27-kDa polypeptide of AMO (20). Importantly, acetylene inactivates AMO only under conditions in which the enzyme is catalytically active (21). Accordingly, if a catalytically inactive pool of AMO is generated by cell lysis, then the majority of AMO should be protected against acetylene inactivation in the absence of exogenously added copper.…”

“…AMO activity in whole cells is sensitive to copper-selective chelating agents, including allylthiourea (14,15,32,33), xanthates (52,53), carbon disulfide (21), ct, ao'-dipyridyl (15), and cyanide (15). Light is also a potent and specific inactivator of AMO (16,42).…”

Bacterial CS2 Hydrolases from Acidithiobacillus thiooxidans Strains Are Homologous to the Archaeal Catenane CS2 Hydrolase

“…CS 2 is one of the oldest known inhibitors of nitrification (57). The inhibitory action has been attributed to the CS 2 molecule forming a complex with a nucleophilic amino acid close to the active center of AMO, thereby chelating the Cu cofactor from the active center (58,59). If MMO is similarly inhibited by CS 2 , then both methanotrophs and nitrifiers would benefit from an enzyme capable of CS 2 removal.…”

Butyric Acid- and Dimethyl Disulfide-Assimilating Microorganisms in a Biofilter Treating Air Emissions from a Livestock Facility

“…AOB assimilation of DMDS was unexpected because AOB are normally considered autotrophic and also because of the addition of thiourea to the incubations. Thiourea specifically inhibits the enzyme ammonia monooxygenase (19), so our results suggest that the DMDS was taken up by an alternative metabolism. The finding of AOB assimilating DMDS carbon could also imply cross-feeding.…”