COMMENTARY COMMENTARY

Is there a pathway for N2Oproductionfrom hydroxylamine oxidoreductase in ammonia-oxidizing bacteria? Corey J. Whitea,b and Nicolai Lehnerta,b,1

- The nitrogen cycle is a key biogeochemical cycle on NO2 back down to N2 during anaerobic respiration, pro- Earth, as nitrogen is an essential nutrient required for all ducing nitric oxide (NO) and nitrous oxide (N2O) in in- life-forms. The largest source of bioavailable nitrogen termediate steps (3).

originates from biological and synthetic nitrogen (N2) Release of both NO and N2O during these transfor- fixation, whereby N2 is converted to ammonia (NH3), mations is of global consequence, as NO participates in which can then be incorporated into biomass the depletion of the ozone layer, whereas N2Ohasbe- by plants. In the developing world, soils often do come the third most significant greenhouse gas, with a

not contain enough nitrogen to give large crop yields, global warming potential that is 300 times that of CO2 (4). so fertilizers, produced by synthetic nitrogen fixation, are Withtheincreaseintheuseoffertilizer since the preindus-

critically important to supplement this lack of nitrogen. trial era, global N2O emissions have increased substan- On the other hand, in the developed world, overfertili- tially. Understanding the processes that generate N2O zation is a common problem (1). In the soil, NH3-oxidiz- from fertilizer in soil and seawater is therefore essential ing bacteria (AOB) and archaea compete for the uptake in devising practical strategies to minimize its production.

of NH3 with plants, aerobically oxidizing it to nitrite AOBs are of particular interest in this regard, as - - (NO2) or nitrate (NO3). The first step of this process they are capable not only of nitrification but also of is catalyzed by ammonia monooxygenase, producing denitrification, termed nitrifier denitrification. These

hydroxylamine (NH2OH). Hydroxylamine oxidoreduc- AOBs have been implicated as significant contributors - tase (HAO) then further oxidizes NH2OH to NO2 (2). in increased N2O emissions (5, 6). Previous work has These oxidation reactions provide soil microbes with re- found that AOBs express nitrite and nitrous oxide re- ducing equivalents for ATP synthesis. Complementary ductases under anaerobic conditions, protecting - to this process is denitrification, which ultimately reduces against NO2 accumulation, and producing N2Oasa direct product (7). Other studies have implicated aer-

obic NH2OH oxidation as another source of trace amounts of NO and N2O (8). Typically, NH2OH is oxi- - dized to NO2 by HAOs, as mentioned above. These enzymes contain a catalytic heme P460 cofactor in the active site. It has been postulated that HAOs could in-

completely oxidize NH2OH to HNO, which, after release from the active site, could then dimerize and dehydrate

to form N2OandH2O. NO is another potential incom- plete oxidation product of NH2OH, which could serve as a substrate for denitrification to produce N2O, or escape into the atmosphere (8). Now, work by Lancaster and coworkers (9) has revealed a potential pathway for the

direct production of N2ObyHAOs. To better understand NH2OH oxidation, the Lancas- ter group studied the enzyme cyt P460 from the AOB Fig. 1. PyMOL depiction of the N. europaea cyt P460 active site [Protein Data Bank Nitrosomonas europaea (10). N. europaea cyt P460 ex- (PDB) ID code 2JE2, Left] and of the N. europaea HAO heme P460 active site ists as a 36-kDa homodimer with a c-type heme in the (PDB ID code 4N4N, Right). active site that has an unusual N−C cross-link from

aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055; and bDepartment of Biophysics, University of Michigan, Ann Arbor, MI 48109-1055 Author contributions: C.J.W. and N.L. wrote the paper. The authors declare no conflict of interest. See companion article on page 14704. 1To whom correspondence should be addressed. Email: [email protected].

14474–14476 | PNAS | December 20, 2016 | vol. 113 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1617953114 Downloaded by guest on September 23, 2021 intermediate, which exhibits a Soret band at 455 nm and is EPR-silent. This intermediate accumulates, suggesting that its decay is the rate-limiting step and, thereby, allowing it to be studied in detail. This species reacts with hydroxylamine in the next step of the reaction with a second-order rate constant, −1 −1 kobs(2) = 0.07 mM ·min . − UV/visible data indicate that this intermediate is oxidized by 3e III relative to the Fe −NH2OH adduct. Lancaster and coworkers propose that this species is a diamagnetic ferric NO complex, or {FeNO}6 in the Enemark−Feltham notation (see Fig. 2). This idea was confirmed by independently producing the {FeNO}6 III species by reacting the resting Fe −H2O complex with the Fig. 2. Proposed mechanism of NH2OH oxidation by N. europaea cyt P460. The square brackets indicate that the ferrous product has NO donor PROLI-NONOate [1-(hydroxy-NNO-azoxy)-L-proline] not been directly observed so far. Reproduced from ref. 9. in an NO-shunted pathway. The resulting complex has an iden- tical UV/visible signature to the observed intermediate and was showntobestablebothinsolutionandinthepresenceofexcess ′ the heme 13 mesocarbon to the amine of Lys70 as shown in Fig. 1, oxidant. Importantly, addition of varying concentrations of Left (11). In contrast, the heme P460 cofactor in the active site of NH OH to the {FeNO}6 complex, generated by reaction of the ′ 2 HAOs is doubly cross-linked by Tyr491 at the 5 mesocarbon and enzyme with NO, shows the same second-order decay (0.07 ± α − − the pyrrole -carbon positions (Fig. 1, Right) (12). Despite this 0.01 mM 1·min 1), confirming that the intermediate that accu- structural difference, studies on cyt P460s from other AOBs, mulates in the rate-determining step of cyt P460 catalysis is, in Methylococcus capsulatus and Kuenenia stuttgartiensis,have fact, the {FeNO}6 complex. broadly implicated cyt P460s in NH2OH oxidation (13, 14). Never- To confirm that N2O is the product of the reaction between theless, it still remains an open question whether the results for the 6 III the {FeNO} intermediate and NH2OH, the Fe −H2Ocomplex N. europaea cyt P460 discussed herein can simply be transferred to was reacted with varying concentrations of NO (provided by HAOs. Further studies on HAOs will be necessary to confirm this. PROLI-NONOate) and NH2OH, and the N2Oyieldwasdeter- In their PNAS paper, Lancaster and coworkers (9) report that mined. A clear 1:1 stoichiometry is observed between the NO the aerobic oxidation of NH2OH by cyt P460 does not result in stoi- 6 - added (and, correspondingly, the {FeNO} generated) and N2O chiometric conversion to NO2 but, instead, leads to the production of 15 14/15 - produced. When using isotopically labeled NH2OH, N2O 70% NO2 and 30% N2O. This finding prompted further studies under is produced only in the presence of cyt P460, confirming that the anaerobic conditions that point toward a direct enzymatic pathway N−N coupling reaction occurs between the {Fe14NO}6 complex for stoichiometric N2O production from the cyt P460 catalyzed oxida- 15 and NH2OH. The product of this coupling reaction should tion of NH2OH. N2O analysis of solutions of the enzyme by GC, with be a ferrous complex (see Fig. 2); however, this species was varying concentrations of either NH2OH or the two-electron oxidant never observed directly. Under the reaction conditions used in phenazine methosulfate (PMS), reveals the stoichiometry for this re- this study, this species is rapidly oxidized to FeIII due to the − action to be 2:1 of NH2OH and PMS to N2O, respectively, suggesting 1 presence of excess oxidant (kobs > 1,100 s ), which prevents that the reaction requires two equivalents of NH2OH and four oxidiz- its characterization. ing equivalents to generate one molecule of N2O, Finally, Lancaster and coworkers examined the reversibility of NO binding in the NO shunt by generating the {FeNO}6 complex → + + - + + 2 NH2OH N2O H2O 4e 4H . [1] with one equivalent PROLI-NONOate. Upon exposure to O2,the UV/visible features of the {FeNO}6 complex disappeared while III− - Mechanistic details of this reaction were obtained by UV/visible the Fe H2O signal appeared. Additionally, NO2 is produced as and EPR studies (see Fig. 2). Spectra obtained for the as-isolated the oxidized product. cyt P460 show the heme in the high-spin ferric state with a Soret In summary, the paper by the Lancaster group (9) describes

band at 440 nm and g values of 6.57, 5.09, and 1.97. It is pro- a new, direct pathway of N2O production from cyt P460s via posed that the active site is six-coordinate, with a likely solvent oxidation of NH2OH that has thus far been overlooked in the III− H2O loosely bound at the active site (Fe H2O). Upon addition of field. This result implies that N2OproductionbyAOBsdoes NH2OH to cyt P460, the 440-nm Soret band associated with the not require nitrifier denitrification. In fact, these findings pro- III− Fe H2O resting state shifts to 445 nm, and Q bands broaden vide a rationale for previous reports of increased N2Oproduc- with the formation of an isosbestic point at 438 nm, suggesting a tion under anaerobic conditions and high concentrations of

single-step conversion. EPR of the product revealed a 95% con- NH3, where nitrifier denitrification is not promoted (16). The version to a low-spin species, consistent with the displacement of second-order decay of the {FeNO}6 intermediate suggests

the bound water molecule with the stronger field NH2OH ligand. that the N. europaea cyt P460 may be responsible for detox- The remaining 5% are converted to an off-path and nonproductive ifying high levels of NH2OH. Taking these ideas a step further, it 7 − - species, a ferrous NO complex, or {FeNO} in Enemark Feltham seems possible that NO2 production by HAOs may not actually be notation (15). Because the electronic structures of transition the catalytic function of HAOs under high NH2OH concentrations metal nitrosyls can be ambiguous (3), this notation is useful in but, instead, that N2O is the main product and that the observed - counting electrons of transition metal NO complexes by treating nitrite is a byproduct of NO oxidation to NO2 in the presence of − “ ” the metal NO unit as a single entity. Here, the superscript 7 O2—a potentially game-changing result. These findings prompt corresponds to the sum of iron d- and NO π*-electrons. further studies on HAOs to better understand their catalytic func- - Addition of the oxidant [Ru(NH3)6]Cl3 and excess NH2OH to the tion and to determine how these enzymes regulate NO and NO2 III Fe −NH2OH complex results in the single-step formation of an production. All of these results have dramatic implications for the

White and Lehnert PNAS | December 20, 2016 | vol. 113 | no. 51 | 14475 Downloaded by guest on September 23, 2021 function of HAOs in nitrification, the potentially underestimated NH2OH oxidation seems now essential in designing ways to mini- role of nitrification in N2O production, and the significance of mize N2O release from agricultural soils and wastewater NH2OH in the nitrogen cycle. The mechanistic understanding of treatment plants.

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4 Ravishankara AR, Daniel JS, Portmann RW (2009) Nitrous oxide (N2O): The dominant ozone-depleting substance emitted in the 21st century. Science 326(5949): 123–125. 5 Ni B-J, Peng L, Law Y, Guo J, Yuan Z (2014) Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways. Environ Sci Technol 48(7):3916–3924.

6 Perez-Garcia O, Villas-Boas SG, Swift S, Chandran K, Singhal N (2014) Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic- oxic transition via flux balance analysis of a metabolic network model. Water Res 60:267–277. 7 Beaumont HJE, van Schooten B, Lens SI, Westerhoff HV, van Spanning RJM (2004) Nitrosomonas europaea expresses a nitric oxide reductase during nitrification. J Bacteriol 186(13):4417–4421. 8 Hooper AB, Terry KR (1979) Hydroxylamine oxidoreductase of Nitrosomonas. Production of nitric oxide from hydroxylamine. Biochim Biophys Acta 571(1):12–20. 9 Caranto JD, Vilbert AC, Lancaster KM (2016) Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission. Proc Natl Acad Sci USA 113:14704–14709. 10 Elmore BO, Pearson AR, Wilmot CM, Hooper AB (2006) Expression, purification, crystallization and preliminary X-ray diffraction of a novel Nitrosomonas europaea cytochrome, cytochrome P460. Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 4):395–398. 11 Pearson AR, et al. (2007) The crystal structure of cytochrome P460 of Nitrosomonas europaea reveals a novel cytochrome fold and heme-protein cross-link. Biochemistry 46(28):8340–8349. 12 Igarashi N, Moriyama H, Fujiwara T, Fukumori Y, Tanaka N (1997) The 2.8 Å structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea. Nat Struct Biol 4(4):276–284. 13 Zahn JA, Duncan C, DiSpirito AA (1994) Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath. J Bacteriol 176(19):5879–5887. 14 Maalcke WJ, et al. (2014) Structural basis of biological NO generation by octaheme oxidoreductases. J Biol Chem 289(3):1228–1242. 15 Enemark JH, Feltham RD (1974) Principles of structure, bonding, and reactivity for metal nitrosyl complexes. Coord Chem Rev 13(4):339–406.

16 Law Y, Ni B-J, Lant P, Yuan Z (2012) N2O production rate of an enriched ammonia-oxidising bacteria culture exponentially correlates to its ammonia oxidation rate. Water Res 46(10):3409–3419.

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