Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH₃) oxidation to nitrite (NO₂⁻) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH₃ oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH₂OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH₂OH or an initial oxidation product other than NH₂OH. We now show through combined physiological and stable isotope tracer analyses that NH₂OH is both produced and consumed during the oxidation of NH₃ to NO₂⁻ by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH₂OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH₃ by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH₂OH, the archaea possibly catalyzing NH₂OH oxidation using a novel enzyme complex.Microbial oxidation of ammonia (NH₃) to nitrite (NO₂⁻), the first step in nitrification, plays a central role in the global cycling of nitrogen. Recent studies have established that marine and terrestrial representatives of an abundant group of archaea, now classified as Thaumarchaeota, are autotrophic NH₃ oxidizers (1–5). Despite increasing evidence that ammonia-oxidizing archaea (AOA) generally outnumber ammonia-oxidizing bacteria (AOB), and likely nitrify in most natural environments, very little is known about their physiology or supporting biochemistry (6, 7). Genome sequence analyses have pointed to a unique pathway for NH₃ oxidation, likely using copper as a major redox active metal, and coupled to a variant of the hydroxypropionate/hydroxybutyrate cycle (8). However, the only genome sequence feature that associates the archaeal pathway for NH₃ oxidation with that of the better characterized AOB is a divergent variant of the ammonia monooxygenase (AMO), which may or may not be a functional equivalent of the bacterial AMO. Thus, the supporting biochemistry of a biogeochemically significant group of microorganisms remains unresolved (8, 9).Among the AOB, as represented by the model organism Nitrosomonas europaea, NH₃ is first oxidized to hydroxylamine (NH₂OH) by AMO, an enzyme composed of three subunits encoded by amoC, amoA, and amoB genes (7). NH₂OH is subsequently oxidized to NO₂⁻ by the hydroxylamine oxidoreductase (HAO) (7), a heme-rich enzyme encoded by the hao gene (7). Of the four electrons released from the oxidation of NH₂OH to NO₂⁻, two are transferred to the terminal oxidase for respiratory purposes and two are transferred to AMO for further oxidation of NH₃ (7). Although all available genome sequences for the AOA contain homologs of the bacterial AMO (amoB, amoC, and amoA), there are no obvious homologs of AOB-like HAO, or cytochromes c554 and c_M552 critical for energy conversion in AOB (8–15). Thus, either the product of NH₃ oxidation is not NH₂OH or, alternatively, these phylogenetically deeply branching thaumarchaea use a novel biochemistry for NH₂OH oxidation and electron transfer (8).In an attempt to gain further insights into the biochemistry and physiology of these unique archaeal nitrifiers, we here investigated the role of NH₂OH in Nitrosopumilus maritimus metabolism. These studies were complicated by the extremely oligotrophic character of this organism contributing to very low cell densities in culture (16). To overcome the challenge of working with low cell density cultures of N. maritimus, we established a method to concentrate cells on nylon membrane filters such that the cells remained competent for NH₃-dependent NO₂⁻ formation and oxygen (O₂) uptake. This method enabled us to carry out relatively short duration physiological studies and stable isotope tracer experiments directed at determining if N. maritimus can oxidize exogenous NH₂OH to NO₂⁻ while consuming O₂ and producing ATP, and if NH₂OH is an intermediate in NH₃ oxidation pathway of N. maritimus.