Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay.

Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor.     

Active site comparison of CoII blue and green nitrite reductases

Copper-containing nitrite reductases (NiRs) possess type 1 (T1) and type 2 (T2) copper sites and can be either green or blue in color owing to differences at their T1 centers. The active sites of a green and a blue NiR were studied by utilizing their T1CuI/T2CoII and T1CoII/T2CoII-substituted forms. The UV/Vis spectra of these derivatives highlight the similarity of the T2 centers in these enzymes and that T1 site differences are also present in the CoII forms. The paramagnetic NMR spectra of T1CuI/T2CoII enzymes allow hyperfine shifted resonances from the three T2 His ligands to be assigned: these exhibit remarkably similar positions in the spectra of both NiRs, emphasizing the homology of the T2 centers. The addition of nitrite results in subtle alterations in the paramagnetic NMR spectra of the T1CuI/T2CoII forms at pH<7, which indicate a geometry change upon the binding of substrate. Shifted resonances from all of the T1 site ligands have been assigned and the CoII--N(His) interactions are alike, whereas the CbetaH proton resonances of the Cys ligand exhibit subtle chemical shift differences in the blue and green NiRs. The strength of the axial CoII--S(Met) interaction is similar in the two NiRs studied, but the altered conformation of the side chain of this ligand results in a dramatically different chemical shift pattern for the CgammaH protons. This indicates an alteration in the bonding of the axial ligand in these derivatives, which could be influential in the CuII proteins.     

Binding and activation of nitrite and nitric oxide by copper nitrite reductase and corresponding model complexes

The reduction of nitrite to nitric oxide in dissimilatory denitrification is carried out by copper nitrite reductases (CuNIRs) via a type 2 copper site. Extended studies on CuNIRs in combination with model complexes have allowed for the establishment of two potential mechanisms for this transformation. Recent experimental and computational results have revealed further details of this process. In addition, the interaction of NO with copper sites has recently gained much attention. This review discusses recent results in the context of the known coordination chemistry of CuNIRs.     

Effect of a tridentate ligand on the structure, electronic structure, and reactivity of the copper(I) nitrite complex: role of the conserved three-histidine ligand environment of the type-2 copper site in copper-containing nitrite reductases

It is postulated that the copper(I) nitrite complex is a key reaction intermediate of copper containing nitrite reductases (Cu-NiRs), which catalyze the reduction of nitrite to nitric oxide (NO) gas in bacterial denitrification. To investigate the structure-function relationship of Cu-NiR, we prepared five new copper(I) nitrite complexes with sterically hindered tris(4-imidazolyl)carbinols [Et-TIC = tris(1-methyl-2-ethyl-4-imidazolyl)carbinol and iPr-TIC = tris(1-methyl-2-isopropyl-4-imidazolyl)carbinol] or tris(1-pyrazolyl)methanes [Me-TPM = tris(3,5-dimethyl-1-pyrazolyl)methane; Et-TPM = tris(3,5-diethyl-1-pyrazolyl)methane; and iPr-TPM = tris(3,5-diisopropyl-1-pyrazolyl)methane]. The X-ray crystal structures of all of these copper(I) nitrite complexes were mononuclear eta(1)-N-bound nitrite complexes with a distorted tetrahedral geometry. The electronic structures of the complexes were investigated by absorption, magnetic circular dichroism (MCD), NMR, and vibrational spectroscopy. All of these complexes are good functional models of Cu-NiR that form NO and copper(II) acetate complexes well from reactions with acetic acid under anaerobic conditions. A comparison of the reactivity of these complexes, including previously reported (iPr-TACN)Cu(NO2) [iPr-TACN = 1,4,7-triisopropyl-1,4,7-triazacyclononane], clearly shows the drastic effects of the tridentate ligand on Cu-NiR activity. The copper(I) nitrite complex with the Et-TIC ligand, which is similar to the highly conserved three-histidine ((His)3) ligand environment in the catalytic site of Cu-NiR, had the highest Cu-NiR activity. This result suggests that the (His)3 ligand environment is essential for acceleration of the Cu-NiR reaction. The highest Cu-NiR activity for the Et-TIC complex can be explained by the structural and spectroscopic characterizations and the molecular orbital calculations presented in this paper. Based on these results, the functional role of the (His)3 ligand environment in Cu-NiR is discussed.     

Redox-state-dependent complex formation between pseudoazurin and nitrite reductase

Bacterial copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide as part of the denitrification process. Pseudoazurin interacts with nitrite reductase in a transient fashion to supply the necessary electrons. The redox-state dependence of complex formation between pseudoazurin and nitrite reductase was studied by nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. Binding of pseudoazurin in the reduced state is characterized by the presence of two binding modes, a slow and a fast exchange mode, with a K(d)(app) of 100 microM. In the oxidized state of pseudoazurin, binding occurs in a single fast exchange mode with a similar affinity. Metal-substituted proteins have been used to show that the mode of binding of pseudoazurin is independent of the metal charge of nitrite reductase. Contrary to what was found for other cupredoxins, protonation of the exposed His ligand to the copper of pseudoazurin, His81, does not appear to be involved directly in the dual binding mode of the reduced form. A model assuming the presence of a minor form of pseudoazurin is proposed to explain the behavior of the complex in the reduced state.     

Nitrite reduction by copper through ligand-mediated proton and electron transfer

Nitrite reduction by a copper complex featuring a proton-responsive tripodal ligand is demonstrated. Gaseous nitric oxide was confirmed as the sole NO_X by-product in quantitative yield. DFT calculations predict that nitrite reduction occurs via a proton and electron transfer process mediated by the ligand. The reported mechanism parallels nitrite reduction by copper nitrite reductase.     

Evaluation of the catalytic mechanism of AICAR transformylase by pH-dependent kinetics, mutagenesis, and quantum chemical calculations.

The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pK(a) of the imidazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type without changes in substrate and cofactor Km values. The kcat-pH profile indicated virtually no pH-dependence in the pH range 6-10.5. The results suggest that the ammonium moiety of Lys or Arg is important in catalysis, most likely acting as a general acid catalyst with a pK(a) value greater than 10.5. The H267A mutant was also prepared since His267 has been found in the active site and implicated in catalysis. The mutant enzyme showed no detectable activity while retaining its binding affinity for substrate, indicating that it plays a critical role in catalysis. We propose that His267 interacts with Lys266 to aid in the precise positioning of the general acid catalyst to the N3 of the imidazole of AICAR.     

Mapping of the binding site on pseudoazurin in the transient 152 kDa complex with nitrite reductase

Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitrite oxide as a part of the denitrification process. In Alcaligenes faecalis S-6, the copper protein pseudoazurin acts as electron donor to NiR. The binding surface of pseudoazurin involved in the formation of the 152 kDa complex with NiR has been determined by NMR using cross saturation from NiR to perdeuterated pseudoazurin. Due to the transient nature of the complex, saturation effects can be observed on the resonances of the unbound protein. The binding site comprises the hydrophobic area surrounding the exposed copper ligand His81, suggesting that this residue is important for efficient electron transfer.     

Molten sodium nitrite—potassium nitrite eutectic: the reaction of some compounds of molybdenum

Molybdenum(VI) oxide, ammonium molybdate and molybdic acid reacted in molten sodium nitrite—potassium nitrite eutectic to form orthomolybdate, nitrogen dioxide and nitric oxide (with nitrate as a secondary product), a more polymerised polymolybdate being formed as an intermediate product. Tungsten(VI) oxide reacted similarly but less rapidly. Molybdenum and tungsten metals reacted to form the orthoxyanion and nitrogen, the latter metal reacting considerably faster and forming smaller amounts of nitric oxide and nitrous oxide. Reaction temperatures and stoichiometries are given and reaction pathways suggested.     

Reduction of nitrite to NO in an organised triphasic medium by platinum carbonyl clusters and redox active dyes as electron carriers

Artificial electron donors such as leuco methylene blue and leuco safranin O reduce nitrite ion to nitric oxide. The reaction is effected in a U-tube where nitrite ion and dye in two aqueous layers are separated by a layer of dichloromethane (a close model for a biological liquid membrane) that contains the platinum carbonyl cluster ([Bu(4)N]2[Pt12(CO)24], Chini cluster). On passing dihydrogen an electron transfer chain involving dihydrogen, the dye, the clusters and the nitrite ion is initiated. The cluster catalytically reduces the dye in the presence of dihydrogen, the reduced dye migrates across the phase boundaries and in turn reduces the nitrite ions. The resultant nitric oxide in the effluent gas has been identified by its reactions with cobalamine and myoglobin. When safranin O is the dye, an adduct is formed between the reduced dye and NO. It has been identified by spectroscopic techniques and its probable structure investigated by DFT calculations.     

The importance of the long type 1 copper-binding loop of nitrite reductase for structure and function

The long 15-residue type 1 copper-binding loop of nitrite reductase has been replaced with that from the cupredoxin amicyanin (7 residues). This sizable loop contraction does not have a significant effect on the spectroscopy, and therefore, the structures of both the type 1 and type 2 Cu(II) sites. The crystal structure of this variant with Zn(II) at both the type 1 and type 2 sites has been determined. The coordination geometry of the type 2 site is almost identical to that found in the wild-type protein. However, the structure of the type 1 centre changes significantly upon metal substitution, which is an unusual feature for this class of site. The positions of most of the coordinating residues are altered of which the largest difference was observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. Flexibility has been introduced into this region of the protein. The 200 mV increase in the reduction potential of the type 1 copper site indicates that structural changes upon reduction must stabilise the cuprous form. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity. The extended type 1 copper-binding loop of this enzyme makes a number of interactions that are important for maintaining quaternary structure.     

Electrocatalytic reductions of nitrite, nitric oxide, and nitrous oxide by thermophilic cytochrome P450 CYP119 in film-modified electrodes and an analytical comparison of its catalytic activities with myoglobin

Previous investigations of nitrite and nitric oxide reduction by myoglobin in surfactant film modified electrodes characterized several distinct steps in the denitrification pathway, including isolation of a nitroxyl adduct similar to that proposed in the P450nor catalytic cycle. To investigate the effect of the axial ligand on these biomimetic reductions, we report here a comparison of the electrocatalytic activity of myoglobin (Mb) with a thermophilic cytochrome P450 CYP119. Electrocatalytic nitrite reduction by CYP119 is very similar to that by Mb: two catalytic waves at analogous potentials are observed, the first corresponding to the reduction of nitric oxide, the second to the production of ammonia. CYP119 is a much more selective catalyst, giving almost exclusively ammonia during the initial half-hour of reductive electrolysis of nitrite. More careful investigations of specific steps in the catalytic cycle show comparable rates of nitrite dehydration and almost identical potentials and lifetimes for ferrous nitroxyl intermediate (Fe(II)-NO(-)) in CYP119 and Mb. The catalytic efficiency of nitric oxide reduction is reduced for CYP119 as compared to Mb, attributable to both a lower affinity of the protein for NO and a decreased rate of N-N coupling. Isotopic labeling studies show ammonia incorporation into nitrous oxide produced during nitrite reduction, as has been termed co-denitrification for certain bacterial and fungal nitrite reductases. Mb has a much higher co-denitrification activity than CYP119. Conversely, CYP119 is shown to be slightly more efficient at the two-electron reduction of N(2)O to N(2). These results suggest that thiolate ligation does not significantly alter the catalytic reactivity, but the dramatic difference in product distribution may suggest an important role for protein stability in the selectivity of biocatalysts.     

Aspects of the mechanism of catalysis of glucose oxidase: A docking, molecular mechanics and quantum chemical study

The complex structure of glucose oxidase (GOX) with the substrate glucose was determined using a docking algorithm and subsequent molecular dynamics simulations. Semiempirical quantum chemical calculations were used to investigate the role of the enzyme and FAD co-enzyme in the catalytic oxidation of glucose. On the basis of a small active site model, substrate binding residues were determined and heats of formation were computed for the enzyme substrate complex and different potential products of the reductive half reaction. The influence of the protein environment on the active site model was estimated with a point charge model using a mixed QM/MM method. Solvent effects were estimated with a continuum model. Possible modes of action are presented in relation to experimental data and discussed with respect to related enzymes. The calculations indicate that the redox reaction of GOX differs from the corresponding reaction of free flavins as a consequence of the protein environment. One of the active site histidines is involved in substrate binding and stabilization of potential intermediates, whereas the second histidine is a proton acceptor. The former one, being conserved in a series of oxidoreductases, is also involved in the stabilization of a C4a-hydroperoxy dihydroflavin in the course of the oxidative half reaction.     

Ion chromatography of nitrite at the ppb level with photometric measurement of iodine formed by post-column reaction of nitrite with iodide

The difficulty in ion-chromatographic determination of nitrite in aqueous solutions containing a high concentration of chloride arises mainly from incomplete resolution of the peaks for these anions on the separation column whose efficiency is not high. A photometric measurement of iodine formed by a reaction of nitrite with iodide has been found to make it possible to determine, chromatographically, trace amounts of nitrite without any interference from chloride; chloride does not oxidize iodide to produce iodine. The proposed method was based on the separation of nitrite from matrix anions on a silica-based anion-exchange column with a 1.5·10⁻³ M phthalate eluent (pH 5.0), followed by photometric measurement of the iodine (as triiodide) formed via a post-column reaction of the separated nitrite with iodide. The optimal conditions for the post-column reaction were established by varying the concentrations of iodide, copper(II) and nitric acid in a post-column-reaction solution and the length of a reaction tube. A calibration graph for nitrite, plotted as peak heights versus concentrations, was linear up to 1.50·10⁻⁵ M (690 ppb). The detection limit, defined at S/N=3, was 1.00·10⁻⁷ M (4.60 ppb) nitrite. The presence of chloride ions up to 0.01 M did not give any interference to the determination of nitrite. This method was successfully applied to the determination of nitrite in lake water, river water, sewage works water and snow samples without any pretreatment.     

Intramolecular electron transfer in nitrite reductases.

The copper- and heme-containing nitrite reductases (NiRs) are key enzymes in denitrification. Their subunits contain two distinct redox-active metal centers, an electron-accepting site and a nitrite-reducing site, to carry out the single-electron reduction of nitrite to nitric oxide. Catalytic cycles of both enzyme families employ intramolecular electron transfer that can be rate-determining for their activity. Herein, we report results comparing these two enzyme families in order to resolve the different mechanisms controlling intramolecular electron transfer in these proteins.     

The binding of nitric oxide at the Cu(i) site of copper nitrite reductase and of inorganic models: DFT calculations of the energetics and EPR parameters of side-on and end-on structures

Density functional theory calculations have been used to probe the end-on and side-on bonding motifs of nitric oxide at the Cu(i) centre in the enzyme copper nitrite reductase and in three inorganic model systems. We find that irrespective of a range of functionals used, the end-on structure is preferred by up to 40 kJ mol(-1), although this preference is smaller for the enzyme than for the inorganic model systems. We have calculated the g-tensor and atomic hyperfine coupling constants for these structures. When compared to available experimental data, for one model compound the calculated EPR parameters definitely favour an end-on structure, although this preference is somewhat less for the enzyme. Our prediction of NO end-on binding in the enzyme is at variance with structural data.     

Protein film voltammetry of copper-containing nitrite reductase reveals reversible inactivation

The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of approximately 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.     

Pulsed ENDOR determination of the arginine location in the ferrous-NO form of neuronal NOS

Mammalian nitric oxide synthases (NOSs) are enzymes responsible for oxidation of L-arginine (L-Arg) to nitric oxide (NO). Mechanisms of reactions at the catalytic heme site are not well understood, and it is of current interest to study structures of the heme species that activates O(2) and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) was used to probe the position of the l-Arg substrate at the NO(?)-coordinated ferrous heme center(s) in the oxygenase domain of rat neuronal NOS (nNOS). The analysis of (2)H and (15)N ENDOR spectra of samples containing d(7)- or guanidino-(15)N(2) labeled L-Arg has resulted in distance estimates for the nearby guanidino nitrogen and the nearby proton (deuteron) at C(δ). The L-Arg position was found to be noticeably different from that in the X-ray crystal structure of nNOS ferrous-NO complex [Li et al. J. Biol. Inorg. Chem.2006, 11, 753-768], with the nearby guanidino nitrogen being ~0.5 ? closer to, and the nearby H(δ) about 1 ? further from, the NO ligand than in the X-ray structure. The difference might be related to the structural constraints imposed on the protein by the crystal. Importantly, in spite of its closer position, the guanidino nitrogen does not form a hydrogen bond with the NO ligand, as evidenced by the absence of significant isotropic hfi constant for N(g1). This is consistent with the previous reports that it is not the L-Arg substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (NO and O(2)) bound to the heme.     

Linkage isomerism in nitrite reduction by cytochrome cd1 nitrite reductase

Nitrite reduction by cytochrome cd(1) nitrite reductase (cd(1)NIR) is currently accepted to involve coordination of the nitrite nitrogen atom to the ferrous d(1) heme. Here, density functional theory results are reported on the previously unexplored O-binding of nitrite to ferrous and ferric cd(1)NIR. Although the N-isomer (nitro) is energetically favored over the O-nitrite (nitrito), even one single strong hydrogen bond may provide the energy required to put the two isomers on level terms. When hydrogen bonding existent at the cd(1)NIR active site is accounted for in the computational model, the O-nitrite isomer is found to spontaneously protonate and thus yield a ferric-hydroxo species, liberating nitric oxide. An O-nitrite ferrous cd(1)NIR complex appears to be an energetically feasible intermediate for nitrite reduction. O-Coordination would offer an advantage since the end product of nitrite reduction would be a ferric-hydroxo/water complex, rather than the more kinetically inert iron-nitrosyl complex implied by the N-nitrite mechanism.