Nitric Oxide Production from Nitrite Reduction and Hydroxylamine Oxidation by Copper-containing Dissimilatory Nitrite Reductase (NirK) from the Aerobic Ammonia-oxidizing Archaeon, Nitrososphaera viennensis

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2− to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14–15N2O from 15NH2OH and 14NO2−. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Aerobic ammonia oxidation, a rate-limiting step of nitrification, drives the global nitrogen cycle (24,40), which involves aerobic ammonia-oxidizing archaea and bacteria (AOA and AOB, respectively) and complete ammonia oxidizers (comammox) (9,44). Of these, AOA primarily contribute to aerobic ammonia oxidation in natural environments including soil and open ocean (19,31,46). AOA are affiliated with the phylum Thaumarchaeota, which includes phylogenetically and physiologically diverse members (6) and the soil-inhabiting archaeon Nitrososphaera viennensis (41). The biochemistry of aerobic ammonia oxidation by AOA has received a great deal of interest because ammonia oxidation to nitrite (NO 2 -) proceeds in a different manner to that of AOB. AOA oxidize ammonia to hydroxylamine by ammonia monooxygenase (Amo) as well as AOB (43), while hydroxylamine is further oxidized to NO 2by an unidentified enzyme (17). All known AOA genomes lack the gene encoding hydroxylamine dehydrogenase (Hao), and the involvement of a copper-protein complex has been proposed (40,45). In parallel with the oxidation of ammonia to NO 2 -, AOA produce nitric oxide (NO) (22). NO is a key intermediate in AOA cells because this highly reactive molecule is essential for sustaining aerobic ammonia oxidation activity (17,33,36,47). To date, the following 2 pathways have been reported as a source of prokaryotic NO formation: NO 2reduction to NO by copper-containing and cytochrome cd 1 -type dissimilatory nitrite reductases (NirK and NirS, respectively) (38) and NH 2 OH oxidation to NO by hydroxylamine oxidoreductase (Hao) (4,21). Although neither nirS nor hao are found in AOA genomes (6), AOA commonly possess nirK, which is transcribed and expressed during aerobic ammonia oxidation (8,15,20,37). These findings suggest that NirK are involved in NO formation in AOA cells. However, NO 2reduction to NO by AOA NirK has never been demonstrated.
Bacterial NirK have been characterized as homotrimeric enzymes, and each subunit has 2 Cu-binding sites (Type 1 and 2 Cu-binding sites). Type 1 Cu-binding sites receive an electron from an electron donor, such as type 1 Cu proteins (single-domain cupredoxins) and/or cytochrome c, and the electron is then further transferred to a type 2 Cu-binding site that is the catalytic center of NirK (14,25). Bacterial NirK have been classified into 2 phylogenetically distinct groups (class 1 and class 2 groups) based on sequence similarities, and the NirK of the class 1 group contains linker loop and tower loop regions in the amino acid sequence (3). AOA NirK, including Ns. viennensis NirK, are affiliated with a distinct clade of bacterial class 1 and 2 groups (Fig. 1A). Lund et al. (20) reported that AOA NirK may be further classified into several phylogenetic clades showing specific geographic distributions. Ns. viennensis NirK has amino acid residues consistent with those of type 1 and 2 Cu-binding sites (His 106 , His 140 , and His 316 for type 1 Cu-binding sites and His 101 , Cys 141 , His 152 , and Met 157 for type 2 Cu-binding sites) as well as the linker and tower loop regions, whereas the C terminus has unusual extensions of ~26 residues (Fig. 1B). These phylogenetic affiliations of and structural variations in Ns. viennensis NirK raise concerns regarding its enzymatic properties, such as specific enzymatic activity, affinity for NO 2 -, and products of NO 2reduction.
Based on its unique sequence and lack of biochemical information, the purpose of the present study was to charac-terize Ns. viennensis NirK. Prior to the present study, we aimed to isolate Ns. viennensis NirK from a batch culture of Ns. viennensis as a native enzyme. However, the activity of aerobic ammonia oxidation often disappeared when we scaled up the cultures (data not shown). Additionally, a slow growth rate (μ max 0.024 h -1 ) (41) and low biomass concentration in the culture (ca. 10 7~8 cells mL -1 ) further precluded the preparation of the biomass required for protein purification. Since recombinant NirK proteins have been successfully used to previously examine several enzymatic properties (7,16,32), the authors decided to heterologously express Ns. viennensis NirK in Escherichia coli, and investigate its enzymatic properties. The nirK gene located in the Ns. viennensis genome (accession number; CP007536.1) was cloned into the expression vector pCold I (Takara Bio, Shiga, Japan) with the 6×His tag using the Mighty cloning reagent set (Takara Bio), and transformed into E. coli strain BL21(DE3) (Takara Bio). The N-terminal region of Ns. viennensis NirK was predicted to be the signal peptide sequence (Met 1 to Ala 24 ), and nirK without the signal peptide sequence was amplified by PCR using ExTaq polymerase (Takara Bio) and specific forward (5ʹ-GGCATATGGCCCCGACTGGTGTCACTAGACACTAT-3ʹ) and reverse (5ʹ-GGAAGCTTAACCAGAGGTGGTGTTGC CACCGGAGG-3ʹ) oligonucleotide primers. The restriction sites of NdeI and HindIII in the forward and reverse primers above are underlined. Genomic DNA extracted from Ns. viennensis cells (JCM19564) was used as the DNA template for PCR. The constructed plasmid was subjected to Sanger sequencing, and no mutations were found in the sequence. Regarding the expression of the recombinant protein in E. coli cells, the expression culture was aerobically cultivated at 37°C in Luria-Bertani media containing 100 ng μL -1 ampicillin. When the OD 600 of the culture increased to 0.4, the culture was transferred to 15°C and held for 30 min, and protein expression was then induced by adding isopropyl β-D-1thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM. After being incubated at 15°C for 24 h, cells were harvested by centrifugation at 8,500×g at 4°C for 10 min. The harvested cells were suspended in buffer containing 20 mM Tris HCl (pH 8), 200 mM NaCl, and 10% glycerol. The cells were disrupted using a sonifier 250 (Branson) (output 20, duty 20% for 60 s, 6 cycles), and centrifuged at 13,000×g at 4°C for 1 h. The supernatant was recovered as a soluble protein fraction, and the recombinant protein was purified using His-tag affinity chromatography. The recombinant protein was bound to His60 Ni Superflow resin (Takara Bio), and washed with washing buffer containing 20 mM Tris HCl (pH 8), 200 mM NaCl, 10% glycerol, and 20 mM imidazole. The bound recombinant protein was eluted with elution buffer containing 20 mM Tris HCl (pH 8), 200 mM NaCl, 10% glycerol, and 300 mM imidazole. Protein concentrations were measured using the DC-protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as previously described (26), and purity was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel as previously described (28). As shown in Fig. 2A, a single protein band appeared at a molecular mass of 40 kDa, which closely matched the molecular mass deduced from amino acid sequences of the recombinant protein (i.e., 39.7 kDa). The protein band was excised from the gel, and subjected to a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis after in-gel tryptic digestion for protein identification (The detailed methodology is described in the Supplementary text). The MALDI-TOF MS analysis confirmed that the protein band corresponded to Ns. viennensis NirK (Fig. S1). Regarding the reconstitution of Cu-binding sites of the recombinant protein, the purified recombinant protein was dialyzed against buffer containing 20 mM Tris HCl (pH 8), 300 mM NaCl, and 0.5 mM CuSO 4 at 4°C for 57 h. The protein solution was dialyzed again using the above Tris buffer without CuSO 4 at 4°C for 6 h. The dialyzed recombinant protein was concentrated using a Vivaspin column (MWCO; 30 kDa) (GE Healthcare Japan, Tokyo, Japan). The recombinant protein was loaded onto a gel-filtration HiLoad 16/600 Superdex 200 pg column (GE Healthcare) to assess the molecular mass of the recombinant protein, which was 105±1.3 kDa (Fig. 2B). Since the deduced molecular mass of Ns. viennensis NirK was 39.7 kDa, the molecular mass obtained by gel filtration indicated that the recombinant protein forms a homotrimeric structure, similar to canonical NirK.
NirK have been characterized as metalloproteins showing a blue or green color spectrum, and exhibit absorption peaks at approximately 450 and/or 600 nm (3). Bacterial NirK, which belong to the class 1 group, often show a maximum absorption peak at approximately 450 nm, although an exception (Achromobacter xylosoxidans NirK) that shows a peak at 593 nm has been previously reported (16). The purified recombinant protein was pale blue in color, and showed an absorption peak at 590 nm (Fig. 2C). This feature indicated that Ns. viennensis NirK is affiliated with the subgroup of NirK showing a blue color spectrum. The blue or green color spectrum of NirK is derived from a copper atom in the type 1 Cu-binding site (14), while the type 2 Cu-binding site does not contribute to the UV or visible spectrum. The type 2 Cu-binding site shows a characteristic electron spin resonance (ESR) spectrum (7,16); therefore, an ESR analysis was performed using a JES-FA200 spectrometer (JEOL, Tokyo, Japan) to test for the presence of the type 2 Cu-binding site in the recombinant protein. An axial type 2 Cu signal (g // =2.24, A // =18.31 mT, and g ⊥ =2.06) was found in the ESR measurement (Fig. 2D), indicating that the recombinant protein has a type 2 Cu-binding site coordinating with a copper atom. Additionally, we assessed the copper content of the recombinant protein by inductively coupled plasma mass spectrometry (ICP-MS). The copper content was found to be 2.9 atoms per subunit of the recombinant protein, indicating that Cu was fully incorporated into the recombinant protein. Overall, the recombinant protein shared the structural and spectroscopic features of class 1 and 2 bacterial NirK, which is consistent with sequencing information.
The kinetics of NO 2reduction were examined by anoxically incubating the recombinant protein at 25°C and pH 6.5 with 15 NO 2and artificial electron donors as previously described (7). All of the buffers and stock solutions were prepared anoxically as previously described (27). Two milliliters of reaction buffer (20 mM phosphate buffer, 0.1 to 1.6 mM Na 15 NO 2 -, 0.5 mM benzyl viologen (BV), and 0.24 mM sodium dithionite) was dispensed into a 1-cm sealable quartz cuvette and placed in an anaerobic chamber in which the O 2 concentration was maintained at lower than 1 ppm. BV was used as an artificial electron donor because it has been employed to examine the kinetics of the NO 2reduction of bacterial NirK (7,13). The cuvette was set in a UV-VIS spectrometer UV-2700 (Shimadzu, Kyoto, Japan), and the initial absorbance of the prepared reaction mixture at a wavelength of 550 nm was approximately 2.0. The reaction was initiated by adding the recombinant protein (50 μL containing 250 μg of protein) using a gastight syringe, and the oxidation rate of reduced BV (molecular extinction coefficient, 10.4 mM -1 cm -1 ) (13) was monitored at 550 nm.
The recombinant protein reduced NO 2by oxidizing BV, whereas no significant BV oxidation was found in the cuvette without the recombinant protein. The turnover number and K m value for NO 2reduction by the recombinant protein were 3.1 s -1 and 287 μM, respectively (Table 1), and the turnover number and affinity constant were markedly lower and higher, respectively, than those of other canonical NirK proteins, including those from AOB. The product of NO 2reduction by the recombinant protein was examined using phenazine methosulfate (PMS) as the electron donor instead of BV. When BV was used as the electron donor, NO 2was reduced to NO, and further reduced to ammonia (approx. 60% of consumed 15 NO 2 -) as observed in a previous study in which the NO 2reduction activity of A. xylosoxidans NirK was examined using methyl viologen (MV) as the electron donor (1). BV and MV have low redox potentials (-350 and -440 mV, respectively) (23), resulting in the reduction of NO to NH 3 ; therefore, PMS with a higher redox potential (+80 mV) was used in the present study. The recombinant protein was incubated as described above in a 1.8-mL gas-tight vial with the addition of 0.5 mM PMS and 5 mM ascorbic acid instead of BV and dithionite, and the production of 15 N-labeled gaseous compounds (i.e., N 2 , NO, and N 2 O) in the headspace was examined by gas chromatography mass spectrometry (GC/MS) as previously described (27). The diluted gases of 15-15 N 2 (Cambridge Isotope Laboratories, Tewksbury, MA, USA), 14 NO, and 14-14 N 2 O (GL Science, Tokyo, Japan) were also analyzed to prepare standard curves for quantification. The recombinant protein reduced 15 NO 2with the oxidation of PMS, and 38 and 48% of consumed 15 NO 2were converted to 15 NO and 15-15 N 2 O, respectively. This is direct evidence to show that the recombinant protein is a NO-forming nitrite reductase. We found that the production of 15-15 N 2 O was equal to the production of 15 (35), as previously observed for a sulfide-linked nitrite reductase (34).
Aside from NO 2reduction, NH 2 OH oxidation was also investigated using the recombinant protein because NH 2 OH is produced as an intermediate during aerobic ammonia oxidation by AOA. The kinetics of NH 2 OH oxidation were examined by aerobically incubating the recombinant protein (245 μg mL -1 ) at 30°C and pH 7.5 with 0.5 mM NH 2 OH, with dissolved oxygen being available as an oxidant. The reaction was initiated by the addition of NH 2 OH solution, and the concentration of NH 2 OH was assessed colorimetrically (5). The concentration of H 2 O 2 , which may be produced by the oxidase activity of NirK (12), was also evaluated colorimetrically using horseradish peroxidase (Wako, Osaka, Japan) and 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMBZ) (Dojindo, Kumamoto, Japan) (2). As shown in Fig. S2, the recombinant protein oxidized NH 2 OH with the production of H 2 O 2 . No NH 2 OH oxidation or H 2 O 2 production was observed when the incubation was repeated without the addition of the recombinant protein.
The values for the turnover number and affinity constant for NH 2 OH oxidation were 0.039 s -1 and 97 μM (Table 1), respectively, and the value for the turnover number was two orders of magnitude lower than that observed for NO 2reduction; therefore, the recombinant protein catalyzed NO 2reduction more efficiently. The addition of cytochrome c from equine heart (1 mg mL -1 ) or BV (0.5 mM) did not result in an increase in the reaction rate or affinity for NH 2 OH oxidation. The product of NH 2 OH oxidation by the recombinant protein was examined in a 15 NH 2 OH tracer experiment (29). The recombinant protein was incubated in a 1.8-mL gas-tight vial with the addition of 0.5 mM 15 NH 2 OH (Cambridge Isotope Laboratories) instead of 14 NH 2 OH. After a 2-h incubation, the concentrations of the 15 N-labeled gaseous products were assessed by GC/MS. The recombinant protein oxidized 15 NH 2 OH and produced 15 NO, 15-15 N 2 O, and 15-15 N 2 gases quantitatively (Fig. 3), whereas the production of NO 2and NH 3 was not detectable (detection limits: 50 and 100 μM, respectively). The oxidation of NH 2 OH to NO has been We repeated the above incubation with the addition of NH 2 OH and NO 2because both compounds are available in AOA cells during aerobic ammonia oxidation. Therefore, the above incubation was repeated with the addition of 15 NH 2 OH and 14 NO 2 -(each 0.5 mM) or 14 NH 2 OH and 15 NO 2 -(Cambridge Isotope Laboratories) (each 0.5 mM). In both cases, 14-15 N 2 O was the major product ( Fig. 3), indicating that the recombinant protein produces N 2 O by oxidizing NH 2 OH using NO 2as an electron acceptor. N 2 O production by the denitrifier NirK from NH 2 OH and NO 2has been previously described (10), and the N-nitrosation reaction is involved in N 2 O production (39). Notably, Ns. viennensis cells produce N 2 O when they are incubated aerobically with NH 3 and NO 2 -(42), although the Ns. viennensis genome lacks the gene encoding nitric oxide reductase (nor) that is involved in N 2 O production from nitrifier-denitrification. Stieglmeier et al. (42) suggested the involvement of Ns. viennensis NirK in the production of N 2 O in an Ns. viennensis culture, and our results support this hypothesis. Although the catalytic efficiency of Ns. viennensis NirK for NH 2 OH oxidation was markedly lower than that of NO 2reduction (Table 1), Ns. viennensis NirK may act as an NH 2 OH oxidase in Ns. viennensis cells and produce N 2 O under oxic growth conditions. Aside from 14-15 N 2 O production, the production of 15 NO and 15-15 N 2 O was also observed when the recombinant protein was incubated with 14 NH 2 OH and 15 NO 2 - (Fig. 3).
Although the recombinant protein catalyzes NO 2reduction and NH 2 OH oxidation, the catalytic efficiency of both reactions was low, as shown in Table 1. AOA nirK transcripts are abundant in the transcriptome (8,11,20,37), suggesting the strong expression of AOA NirK in cells. NirK was the 225 th most abundant protein of the 1,503 proteins detected in the proteome of the late exponential phase of Ns. viennensis cells aerobically oxidizing ammonia (15). The strong expression of NirK appears to support the activity of NO 2reduction to NO as well as NH 2 OH oxidation to NO by the low efficiency catalytic enzyme. Ns. viennensis NirK may function as a bifunctional enzyme that supplies NO molecules from 2 different sources (i.e., NH 2 OH and NO 2 -), which provides Ns. viennensis cells with a competitive advantage. In the present study, the enzymatic kinetics of recombinant Ns. viennensis NirK for NO 2reduction were examined using artificial electron donors; further studies are needed to identify physiological electron donors in Ns. viennensis cells. Bacterial NirK may accept electrons supplied from single-domain cupredoxin and cytochrome c (14,25). A number of genes encoding singledomain cupredoxin were found in the Ns. viennensis genome (Table S1), whereas the ortholog of the gene encoding cytochrome c was not. To date, the biochemistry of AOA cupredoxin has not been investigated using natural enzymes and recombinant proteins, and our study provides basic information that furthers our understanding of the biochemistry of AOA.