Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from Nitrosomonas europaea

Abstract

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g(parallel) = 2.24, A(parallel) = 18.4 mT and g(perpendicular) = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g(parallel) = 2.29, A(parallel) = 16.1 mT and g(perpendicular) = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu(2+) and three diamagnetic Cu(1+) per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per alphabetagamma-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe(3+) was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu(2+)-EPR signal.

Integrated structural biology and molecular ecology of N‐cycling enzymes from ammonia‐oxidizing archaea

“…This result is perhaps not surprising, given that the archaeal AmoB crystal structure lacks one of the three active site histidine residues required to stably coordinate dicopper (Fig. C), a known and necessary cofactor in bacterial ammonia oxidation (Balasubramanian et al ., ; Gilch et al ., ). While this third histidine is present in the archaeal sequence, it is disordered in the crystal structure, perhaps due to a lack of potentially stabilizing interactions provided by the missing domain.…”

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

“…The initial oxidation of ammonia is catalyzed by membrane-bound AMO while hydroxylamine (NH 2 OH), the immediate product of ammonia oxidation, is further oxidized to nitrite (NO 2 Ϫ ) by the periplasmic enzyme hydroxylamine dehydrogenase (HAO) (5). Although N. europaea is the most extensively studied AOB, studies of AMO in this bacterium and AOB in general have historically been hampered by the labile nature of this enzyme (6)(7)(8). However, even though AMO has not yet been obtained in a highly purified active state, considerable insights into the activities and structure of this important enzyme have been obtained from whole-cell studies of N. europaea using different classes of inhibitors (5,9).…”

Application of an Integrated Statistical Design for Optimization of Culture Condition for Ammonium Removal by Nitrosomonas europaea

“…Moreover, metallic ions can lead to the reprogramming of cellular metabolic network during fermentation. The addition of Mg 2+ [16], Cu 2+ and Fe 3+ [5] is found to increase the activity of the ammonia oxidation.…”

“…Among all the microorganisms listed as good degrader of ammonium, Nitrosomonas europaea , the model chemolithoautotrophic ammonium oxidizing bacterium (AOB), obtains energy from the aerobic oxidation of ammonia (NH 3 ) to nitrite (NO 2 − ) [4]. Carbon dioxide (CO 2 ) was used as the preferred assimilative carbon source via the Calvin-Benson-Bassham cycle for N.europaea ’s growth and maintenance [5]. A significant amount of the energy obtained from oxidation of hydroxylamine has to be invested in ammonia oxidation to hydroxylamine, reverse electron transport to generate Nicotinamide adenine dinucleotide (NADH) and CO 2 fixation.…”