Copper-containing Nitrite Reductase Research Articles

Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome

Nitrosomonas europaea, an aerobic ammonia oxidizing bacterium, is responsible for the first and rate-limiting step of the nitrification process, and their ammonia oxidation activities are critical for the biogeochemical cycling and the biological nitrogen removal of wastewater treatment. In the present study, N.europaea cells were cultivated in the inorganic or organic media (the NBRC829 and the nutrient-rich, NR, media, respectively), and the cells proliferated in the form of planktonic and biofilm in those media, respectively. The N.europaea cells in the biofilm growth mode produced larger amounts of the extracellular polymeric substances (EPS), and the composition of the EPS was characterized by the chemical analyses including Fourier transform infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (NMR) measurements. The RNA-Seq analysis of N.europaea in the biofilm or planktonic growth mode revealed that the following gene transcripts involved in central nitrogen metabolisms were abundant in the biofilm growth mode; amo encoding ammonia monooxygenase, hao encoding hydroxylamine dehydrogenase, the gene encoding nitrosocyanine, nirK encoding copper-containing nitrite reductase. Additionally, the transcripts of the pepA and wza involved in the bacterial floc formation and the translocation of EPS, respectively, were also abundant in the biofilm-growth mode. Our study was first to characterize the EPS production and transcriptome of N.europaea in the biofilm and planktonic growth mode.

Biochemical Characterization of the Copper Nitrite Reductase from Neisseria gonorrhoeae.

The copper-containing nitrite reductase from Neisseria gonorrhoeae has been shown to play a critical role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with a type 1 copper center per subunit and a type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized for the first time by EPR and CD spectroscopy, showing that the type 1 copper center has a high rhombicity due to its lower symmetry and more tetragonal structure, while the type 2 copper center has the usual properties, but with a smaller hyperfine coupling constant (A// = 10.5 mT). The thermostability of the enzyme was analyzed by differential scanning calorimetry, which shows a single endothermic transition in the thermogram, with a maximum at 94 °C, while the CD spectra in the visible region indicate the presence of the type 1 copper center up to 80 °C. The reoxidation of the N. gonorrhoeae copper-containing nitrite reductase in the presence of nitrite were analyzed by visible spectroscopy and showed a pH dependence, being higher at pH 5.5-6.0. The high thermostability of this enzyme may be important to maintaining a high activity in the extracellular space and to making it less susceptible to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.

Optimising Electrical Interfacing between the Trimeric Copper Nitrite Reductase and Carbon Nanotubes.

The immobilization of copper-containing nitrite reductase (NiR) from Alcaligenes faecalis on functionalised multi-walled carbon nanotube (MWCNT) electrodes is reported. It is demonstrated that this immobilization is mainly driven by hydrophobic interactions, promoted by the modification of MWCNTs with adamantyl groups. Direct electrochemistry shows high bioelectrochemical reduction of nitrite at the redox potential of NiR with high current density of 1.41 mA cm-2 . Furthermore, the desymmetrization of the trimer upon immobilization induces an independent electrocatalytic behavior for each of the three enzyme subunits, corroborated by an electron-tunneling distance dependence.

Identification of Fungal and Bacterial Denitrification Inhibitors Targeting Copper Nitrite Reductase.

The usage of nitrification inhibitors is one of the strategies that reduce or slow down the denitrification process to prevent nitrogen loss to the atmosphere in the form of N2O. Directly targeting microbial denitrification could be one of the mitigation strategies; however, until now little efforts have been devoted toward the development of denitrification inhibitors. Here, we have identified small-molecule inhibitors of one of the proteins involved in the fungal denitrification pathway. Specifically, virtual screening was employed to identify the inhibitors of copper-containing nitrite reductase (FoNirK) of the filamentous fungus Fusarium oxysporum. Three series of chemical compounds were identified, out of which compounds belonging to two chemical scaffolds inhibited FoNirK enzymatic activity in low micromolar ranges. Several compounds also displayed moderate inhibition of fungal denitrification activity in vivo. Evaluation of in vitro activity against NirK from denitrifying bacterium Achromobacter xylosoxidans (AxNirK) and in vivo bacterial denitrification revealed a similar inhibitory profile.

Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a ∼ 100-fold lower turnover number compared to the wild type enzyme. UV–Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

A molecular dynamics and quantum mechanical investigation of intermolecular interaction and electron-transfer mechanism between copper-containing nitrite reductase and redox partner pseudoazurin.

Much of biological electron transfer occurs between proteins. These molecular processes usually involve molecular recognition and intermolecular electron transfer (inter-ET). The inter-ET reaction between copper-containing nitrite reductase (CuNiR) and partner protein pseudoazurin (PAz) is the first step in denitrification, which is affected by intermolecular association. However, the transient interaction between CuNiR and PAz and the indistinct inter-ET pathway pose challenges for people to understand the biological functions of the CuNiR-PAz complex. Thus, molecular dynamics simulation and quantum mechanical calculation were used to investigate the question in this study. The interaction of the interface residues was determined through hydrogen bonds, root-mean-square deviation, root-mean-square fluctuation, the dynamics cross-correlation matrix, and molecular mechanics Poisson-Boltzmann surface area of molecular dynamics simulations. The interactions among the residues Glu89, Gly200, Asp205, Asn91, Glu204, Thr92, and Met141 on CuNiR and the residues Lys109, Ala15, Lys10, Asn9, Ile110, Met84, and Met16 on PAz are responsible for the stabilization of the complex. The binding free energy is up to -25.33 kcal mol-1. We compared the wild-type and mutant (M84A) interfacial optimized complex models at the CAM-B3LYP level with Grimme dispersion corrections (GD3) to confirm Met84 as a relay station for promoting the inter-ET. Additionally, to test whether Met84 may combine with the adjacent Met141 to form a special two-center, three-electron (S∴S)+ structure to promote the inter-ET, QM/MM was further performed to discuss the possibility of generating an electron stepping stone. Our study will promote a deep understanding of the stable protein-protein interaction, and the identified inter-residue interaction will be theoretical guidance for enhancing the catalytic activity of CuNiR in denitrification.

Differential responding patterns of the nirK-type and nirS-type denitrifying bacterial communities to an Ulva prolifera green tide in coastal Qingdao areas

Coastal eutrophication may be a vital inducement of green tide. Denitrification is an important nitrogen removal pathway that involves a series of enzymatic reactions. The rate-limiting step in the conversion of nitrite to nitric oxide is encoded by two functionally equivalent but structurally distinct genes, copper-containing nitrite reductase gene (nirK) and cytochrome cd1-containing nitrite reductase gene (nirS). Here, we used Illumina Miseq sequencing approach to examine the variations in denitrifying bacterial community characteristics and interactions during an Ulva prolifera green tide in coastal Qingdao areas. Our findings suggested that the variations in the denitrifying bacterial community structure during the green tide were closely related to the changes of chlorophyll a content, salinity and dissolved oxygen content. The nirK-type denitrifying bacteria were more sensitive to green tide than the nirS-type denitrifying bacteria. Additionally, the nirK-type denitrifying bacterial interactions were more stable and complex during the outbreak phase, while the nirS-type denitrifying bacterial interactions were more stable and complex during the decline phase. All of these characters demonstrated that the nirK-type and nirS-type denitrifying bacteria respond differently to the green tide, implying that they may occupy different niches during the green tide in coastal Qingdao areas.

A new concept of waste iron recycling for the enhancement of the anammox process

As a by-product of industry, waste iron scraps (WIS) are low-cost and widely available, which was potential for the development of iron-assisted anammox. In this study, the feasibility of adding WIS to enhance the nitrogen removal of the anammox process (also called WIS-assisted anammox) was demonstrated. Results indicated that the WIS-assisted anammox reactors performed a 15–35% higher nitrogen removal efficiency than that of the control. Compared to the sludge from the control, the sludge from the WIS-assisted anammox reactors had a higher iron content (78–113 g kg−1 SS) and a better specific anammox activity (10.8–15.5 mg N g−1 VSS h−1). The enhanced growth of the anammox bacteria (related to Ca. Kuenenia stuttgartiensis with 99% similarity) in the WIS-assisted anammox reactors was also confirmed by high-throughput sequencing and qPCR. Furthermore, the functional genes predicted by PICRUSt2 revealed a higher level of hydroxylamine oxidoreductase (hao)-like proteins expression of the biomass from the WIS-assisted anammox reactors, implying that the hydroxylamine-related anammox pathway was promoted. Additionally, the observation of cytoplasmic nitrate reductase (narG), copper-containing nitrite reductase (nirK), and nitric oxide reductase (norB) suggested that the introduction of WIS might promote the denitrification ability. This was correlated to the lower ΔNO3-/ΔNH4+ ratio observed in these WIS-assisted anammox reactors. Overall, the WIS-assisted anammox offers a sustainable nitrogen removal process for wastewater treatment with waste iron recycling.

Current status of neutron crystallography in structural biology.

Hydrogen atoms and hydration water molecules in proteins are essential for many biochemical processes, especially enzyme catalysis. Neutron crystallography enables direct observation of hydrogen atoms, and reveals molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. The use of neutron crystallography is still limited for proteins, but its popularity is increasing owing to an increase in the number of diffractometers for structural biology at neutron facilities and advances in sample preparation. According to the characteristics of the neutrons, monochromatic or quasi-Laue methods and the time-of-flight method are used in nuclear reactors and pulsed spallation sources, respectively, to collect diffraction data. Growing large crystals is an inevitable problem in neutron crystallography for structural biology, but sample deuteration, especially protein perdeuteration, is effective in reducing background levels, which shortens data collection time and decreases the crystal size required. This review also introduces our recent neutron structure analyses of copper amine oxidase and copper-containing nitrite reductase. The neutron structure of copper amine oxidase gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions. Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified, and these observations are consistent with the results from the quantum chemical calculations. This review article is an extended version of the Japanese article, Elucidation of Enzymatic Reaction Mechanism by Neutron Crystallography, published in SEIBUTSU-BUTSURI Vol. 61, p.216–222 (2021).

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological and pathological events in biology. Metal coordination chemistry, especially with iron, is at the heart of many biological transformations involving NO. A series of heme proteins, nitric oxide synthases (NOS), soluble guanylate cyclase (sGC), and nitrophorins, are responsible for the biosynthesis, sensing, and transport of NO. Alternatively, NO can be generated from nitrite by heme- and copper-containing nitrite reductases (NIRs). The NO-bearing small molecules such as nitrosothiols and dinitrosyl iron complexes (DNICs) can serve as an alternative vehicle for NO storage and transport. Once NO is formed, the rich reaction chemistry of NO leads to a wide variety of biological activities including reduction of NO by heme or non-heme iron-containing NO reductases and protein post-translational modifications by DNICs. Much of our understanding of the reactivity of metal sites in biology with NO and the mechanisms of these transformations has come from the elucidation of the geometric and electronic structures and chemical reactivity of synthetic model systems, in synergy with biochemical and biophysical studies on the relevant proteins themselves. This review focuses on recent advancements from studies on proteins and model complexes that not only have improved our understanding of the biological roles of NO but also have provided foundations for biomedical research and for bio-inspired catalyst design in energy science.

Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin-like mediator.

We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key β-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.

Improvement of fish production and water quality in a recirculating aquaculture pond enhanced with bacteria-microalgae association

Intensive aquaculture plays an essential role in meeting human beings' growing demand for aquatic products. Recirculating pond has been considered as an effective approach for water and energy saving, but its wastewater also causes severely impair to aquatic animal health in some extent due to residual noxious nitrogen compounds (such as ammonia and nitrite) which are derived from mineralisation and break-up of uneaten feed and fish faeces. In order to purify water efficiently, biological processes are combined with recirculating pond such as using biological filtration, adding probiotics and so on. In this study, a field experiment was conducted to evaluate the potential of a closed recirculating aquaculture pond enhanced with bacteria-microalgae association in improving fish production and water quality. The results showed that the concentrations of total nitrogen (TN), total ammonia-N (TAN) and nitrite-N in the group of bacteria-microalgae with biofilm carriers were reduced by 29.15%, 51.28% and 33.48%, respectively, compared with treatment without any bioaugmentation. Meanwhile, the fish production was significantly improved (13.3%). Microbial community analysis (qPCR and high-throughput sequencing of 16 s and 18 s rRNA genes) suggested that the application of bacteria-microalgae with biofilm carries reduced the abundance of chiA gene (83.19%) which convert organic nitrogen into ammonia-N and increased the abundance of ammonia-monooxygenase (amoA) gene carried by ammonia-oxidizing bacteria (330.75%) and copper-containing nitrite reductase (nirK) gene (464.44%), indicating that the conversion of organic nitrogen to inorganic nitrogen was reduced and the denitrification process was increased. At the initial period, most of nitrogenous compounds were assimilated by microalgae. Along with the experiment, the bacteria community relative to nitrification and denitrification was also increased in bacteria-microalgae with biofilm carriers group compared with CK, such as Delftia (500%) and Acidovorax (450%) in ammoxidation process.

Enhancing denitrification in constructed wetland with algae addition.

Constructed wetlands (CWs) can be used for tertiary treatment of wastewater; however, carbon source shortages limit denitrification. We studied the effect of algae addition as an external carbon source in CWs and found that the nitrogen removal efficiency of CWs is highly dependent on the algae dosage. Optimal nitrogen removal percentage (80.5%) can be achieved by adding 81.1 mg·L-1 dry weight algae to the influent when the chemical oxygen demand/nitrogen (COD/N) ratio reaches 5.3. Longitudinal changes in the nitrogen concentrations, organic matter concentrations, and nitrogen functional genes were also analyzed. The algae addition strengthened the anoxic environment, boosted the volatile fatty acid concentrations, and improved the ratio of nitrite reductase gene (nirS) and copper-containing nitrite reductase (nirK)/16S rRNA, as well as the ratio of nitrate reductase gene (narG)/16S rRNA, thereby expanding the active space for denitrification. The addition of algae could potentially provide enough carbon to enhance denitrification during treatment of wastewater with a low COD/N ratio.

An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures.

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br 2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br 2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

Reverse protein engineering of a novel 4-domain copper nitrite reductase reveals functional regulation by protein-protein interaction.

Cu-containing nitrite reductases that convert NO2- to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br2D NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br2D NiR revealing up to ~500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br2D NiR and anengineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br2D NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.

Structures of substrate- and product-bound forms of a multi-domain copper nitrite reductase shed light on the role of domain tethering in protein complexes.

Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitro-gen cycle where nitrate is used in place of di-oxy-gen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd 1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering than the intuitive advantage for a partner-protein electron-transfer complex by narrowing the conformational search in such a combined system.

Unique hexameric structure of copper-containing nitrite reductase of an anammox bacterium KSU-1

Anaerobic ammonium oxidation (anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the anammox reaction—reduction of nitrite to nitric oxide—is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an anammox bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.

Studying Electron Transfer Pathways in Oxidoreductases

Oxidoreductases containing transition metal ions are widespread in nature and are essential for living organisms. The copper-containing nitrite reductase (NirK) and the molybdenum-containing aldehyde oxidoreductase (Aor) are typical examples of oxidoreductases. Metal ions in these enzymes are present either as mononuclear centers or organized into clusters and accomplish two main roles. One of them is to be the active site where the substrate is converted into product, and the other one is to serve as electron transfer center. Both enzymes transiently bind the substrate and an external electron donor/acceptor in NirK/Aor, respectively, at distinct protein points for them to exchange the electrons involved in the redox reaction. Electron exchange occurs through a specific intra-protein chemical pathway that connects the different enzyme metal cofactors. Based on the two oxidoreductases presented here, we describe how the different actors involved in the intra-protein electron transfer process can be characterized and studied employing molecular biology, spectroscopic, electrochemical, and structural techniques.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

Nitrate removal performance and diversity of active denitrifying bacteria in denitrification reactors using poly(L-lactic acid) with enhanced chemical hydrolyzability.

Poly(L-lactic acid) (PLLA) can be used as an external electron donor in denitrification reactors to treat drinking water, aquaculture water, and industrial wastewater with an imbalanced carbon/nitrogen ratio. However, for PLLA to function in these applications, its chemical hydrolyzability requires improvement. Although the adjustment of the crystallinity (Xc) is effective in improving the hydrolyzability of PLLA, the condition for the Xc of PLLA, in which a sufficient amount of lactic acid is released for denitrification, must be clarified. Therefore, this study investigated the effective Xc range and optimal PLLA content as an electron donor for continuous nitrate removal in denitrification reactors. This study also explored the abundance, succession, and diversity of active denitrifying bacteria in denitrification reactors. The nitrate removal activity of activated sludge using the highly crystalline PLLA (Xc = 39.4%) was 1.8 mg NO3- -N g MLSS-1 h-1, which is 2.4 times higher than that using the nearly amorphous PLLA (Xc = 0.9%). During the 57 days of operation, the denitrification reactor with 3% (w/v) highly crystalline PLLA continued to completely remove nitrate, with a maximum nitrate removal activity of 22.8 mg NO3- -N g MLSS-1 h-1. The 16S rRNA amplicon sequencing and clone library analyses are using transcripts of two nitrite reductase genes, encoding cytochrome cd1 nitrite reductase, and copper-containing nitrite reductase revealed that bacteria belonging to the families Comamonadaceae, Rhodocyclaceae, and Alcaligenaceae were active denitrifying bacteria in the denitrification reactor using PLLA.