Copper Nitrite Reductase Research Articles

Nitrite reductase-mimicking catalysis temporally regulating nitric oxide concentration gradient adaptive for antibacterial therapy.

The unique bacterial infection microenvironment (IME) usually requires complicated design of nanomaterials to adapt to IME for enhancing antibacterial therapy. Here, an alternative IME adaptative nitrite reductase-mimicking nanozyme is constructed by in situ growth of ultrasmall copper sulfide clusters on the surface of a nanofibrillar lysozyme assembly (NFLA/CuS NHs), which can temporally regulate nitric oxide (NO) gradient concentration to kill bacteria initially and promote tissue regeneration subsequently. Benefiting from a copper nitrite reductase (CuNIR)-inspired structure with CuS cluster as active center and NFLA as skeleton, NFLA/CuS NHs efficiently boost the catalytic reduction of nitrite to NO. The inherent supramolecular fibrillar networks displays excellent bacterial capture capability, facilitating initial high-concentration NO attacks on the bacteria. The subsequent catalytic release of low-concentration NO by NFLA/CuS NHs-mediated nitrite reduction remarkably promotes cell migration and angiogenesis. This work paves the way for dynamically eliminating MDR bacterial infection and promoting tissue regeneration in a simple and smart way through CuNIR-mimicking catalysis.

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

Revving up a Designed Copper Nitrite Reductase Using Non-Coded Active Site Ligands.

Herein, we report a three stranded coiled-coil (3SCC) de novo protein containing a type II copper center (CuT2) composed of 6-membered ring N-heterocycles. This design yields the most active homogenous copper nitrite reductase (CuNiR) mimic in water. We achieved this result by controlling three factors. First, previous studies with Nδ and Nε -Methyl Histidine had indicated that a ligand providing pyridine-like electronic character to the copper site was superior to the more donating Nδ for nitrite reduction. By substitution of the parent histidine with the non-coded amino acids pyridyl alanine (3'-Pyridine [3'Py] vs 4'-Pyridine [4'Py]), an authentic pyridine donor was employed without the complications of the coupling of both electronic and tautomeric effects of histidine or methylated histidine. Second, by changing the position of the nitrogen atom within the active site (4'-Pyridine vs. 3'Pyridine) a doubling of the enzyme's catalytic efficiency resulted. This effect was driven exclusivity by substrate binding to the copper site. Third, we replaced the leucine layer adjacent to the active site with an alanine, and the disparity between the 3'Py and 4'Py became more apparent. The decreased steric bulk minimally impacted the 3'Py derivative; however, the 4'Py K m decreased by an order of magnitude (600 mM to 50 mM), resulting in a 40-fold enhancement in the k cat/K m compared to the analogues histidine site and a 1500-fold improvement compared with the initially reported CuNiR catalyst of this family, TRIW-H. When combined with XANES/EXAFS data, the relaxing of the Cu(I) site to a more 2-coordinate Cu(I) like structure in the resting state increases the overall catalytic efficiency of nitrite reduction via the lowering of K m. This study illustrates how by combining advanced spectroscopic methods, detailed kinetic analysis, and a broad toolbox of amino acid side chain functionality, one can rationally design systems that optimize biomimetic catalysis.

Spectroscopically validated multiple structures from one crystal (MSOX) and damage-free atomic structures using XFEL for copper nitrite reductases

Spectroscopically validated multiple structures from one crystal (MSOX) and damage-free atomic structures using XFEL for copper nitrite reductases

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.

Elucidation of the Electrocatalytic Nitrite Reduction Mechanism by Bio-Inspired Copper Complexes.

Mononuclear copper complexes relevant to the active site of copper nitrite reductases (CuNiRs) are known to be catalytically active for the reduction of nitrite. Yet, their catalytic mechanism has thus far not been resolved. Here, we provide a complete description of the electrocatalytic nitrite reduction mechanism of a bio-inspired CuNiR catalyst Cu(tmpa) (tmpa = tris(2-pyridylmethyl)amine) in aqueous solution. Through a combination of electrochemical studies, reaction kinetics, and density functional theory (DFT) computations, we show that the protonation steps take place in a stepwise manner and are decoupled from electron transfer. The rate-determining step is a general acid-catalyzed protonation of a copper-ligated nitrous acid (HNO2) species. In view of the growing urge to convert nitrogen-containing compounds, this work provides principal reaction parameters for efficient electrochemical nitrite reduction. This contributes to the investigation and development of nitrite reduction catalysts, which is crucial to restore the biogeochemical nitrogen cycle.

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.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

Conformational triggering in voltammetry and single-molecule conductivity of two-centre redox metalloproteins: Cytochrome c4 and copper nitrite reductase

Blue copper enzymes often show no voltammetry themselves, whereas substrate binding triggers strong electrocatalytic signals. Similarly, electrochemical STM only gives strong contrasts when substrate (O 2 , NO 2 - ) is present. AFM shows that CuNIR on Au(111)-electrodes modified by self-assembled cysteamine monolayers (SAMs) maintains constant height throughout the electrocatalytic range, while NO 2 - triggers substantial enzyme “swelling”. “Swelling” does not accord with the crystalline state, which, however, is not the relevant catalytic environment. With a view on understanding these patterns, we present ab initio quantum chemical studies of CuNIR/OH 2 and CuNIR/NO 2 - 740-atom fragments including the type I and type II Cu-centres. Replacing water at the type II centre by nitrite triggers two-Å Cu-Cu distance increase, according with enzyme “swelling”. 2 Å Cu-Cu increase would close intramolecular ET entirely, but is compensated by efficient superexchange alignment of closely interacting LUMOs and HOMOs. In the water-bound enzyme these orbitals are separated by unfavourable through-space tunneling regions. • Single-crystal voltammetry of cytochrome c 4 and copper nitrite reductase (CuNIR). • Electrochemical STM and AFM of cyt c 4 and CuNIR. • DFT of intramolecular ET in cyt c 4 . • Ab initio quantum chemistry calculations of 740 atoms CuNIR fragment . • Nitrite triggered CuNIR intramolecular ET channel. • Nitrite triggered CuNIR conformational enzyme swelling in AFM.

NO Coupling at Copper to cis-Hyponitrite: N2O Formation via Protonation and H-Atom Transfer.

Copper nitrite reductases (CuNIRs) convert NO2- to NO as well as NO to N2O under high NO flux at a mononuclear type 2 Cu center. While model complexes illustrate N-N coupling from NO that results in symmetric trans-hyponitrite [CuII]-ONNO-[CuII] complexes, we report NO assembly at a single Cu site in the presence of an external reductant Cp*2M (M = Co, Fe) to give the first copper cis-hyponitrites [Cp*2M]{[CuII](κ2-O2N2)[CuI]}. Importantly, the κ1-N-bound [CuI] fragment may be easily removed by the addition of mild Lewis bases such as CNAr or pyridine to form the spectroscopically similar anion {[CuII](κ2-O2N2)}-. The addition of electrophiles such as H+ to these anionic copper(II) cis-hyponitrites leads to N2O generation with the formation of the dicopper(II)-bis-μ-hydroxide [CuII]2(μ-OH)2. One-electron oxidation of the {[CuII](κ2-O2N2)}- core turns on H-atom transfer reactivity, enabling the oxidation of 9,10-dihydroanthracene to anthracene with concomitant formation of N2O and [CuII]2(μ-OH)2. These studies illustrate both the reductive coupling of NO at a single copper center and a way to harness the strong oxidizing power of nitric oxide via the neutral cis-hyponitrite [Cu](κ2-O2N2).

Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.

Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron–electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

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.

Nitrite reductase activity within an antiparallel de novo scaffold.

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

Damage-free structures of green copper nitrite reductase obtained by neutron crystallography and XFEL

Damage-free structures of green copper nitrite reductase obtained by neutron crystallography and XFEL

QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study.

The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment.

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

Homogeneous batch micro-crystallization of proteins from ammonium sulfate.

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to afew micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate α-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.

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.