Mechanisms of ferriheme reduction by nitric oxide: nitrite and general base catalysis

The reductive nitrosylation (Fe(III)(P) + 2NO + H(2)O = Fe(II)(P)(NO) + NO(2)(-) + 2H(+)) of the ferriheme model Fe(III)(TPPS) (TPPS = tetra(4-sulfonatophenyl)porphyrinato) has been investigated in moderately acidic solution. In the absence of added or adventitious nitrite, this reaction displays general base catalysis with several buffers in aqueous solutions. It was also found that the nitrite ion, NO(2)(-), is a catalyst for this reaction. Similar nitrite catalysis was demonstrated for another ferriheme model system Fe(III)(TMPy) (TMPy = meso-tetrakis(N-methyl-4-pyridyl)porphyrinato), and for ferriheme proteins met-hemoglobin (metHb) and met-myoglobin (metMb) in aqueous buffer solutions. Thus, it appears that such catalysis is a general mechanistic route to the reductive nitrosylation products. Two nitrite catalysis mechanisms are proposed. In the first, NO(2)(-) is visualized as operating via nucleophilic addition to the Fe(III)-coordinated NO in a manner similar to the reactions proposed for Fe(III) reduction promoted by other nucleophiles. This would give a labile N(2)O(3) ligand that hydrolyzes to nitrous acid, regenerating the original nitrite. The other proposal is that Fe(III) reduction is effected by direct outer-sphere electron transfer from NO(2)(-) to Fe(III)(P)(NO) to give nitrogen dioxide plus the ferrous nitrosyl complex Fe(II)(P)(NO). The NO(2) thus generated would be trapped by excess NO to give N(2)O(3) and, subsequently, nitrite. It is found that the nitrite catalysis rates are markedly sensitive to the respective Fe(III)(P)(NO) reduction potentials, which is consistent with the behavior expected for an outer-sphere electron-transfer mechanism. Nitrite is the product of NO autoxidation in aqueous solution and is a ubiquitous impurity in experiments where aqueous NO is added to an aerobic system to study biological effects. The present results demonstrate that such an impurity should not be assumed to be innocuous, especially in the context of recent reports that endogenous nitrite may play physiological roles relevant to the interactions of NO and ferriheme proteins.     

Mechanistic studies on the nitrite-catalyzed reductive nitrosylation of highly charged anionic and cationic Fe(III) porphyrin complexes

The nitrosyl complexes formed during the binding of NO to the (Pn)FeIII(H2O)2 (n = 8+ and 8-) complexes, viz., (P8-)FeII(H2O)(NO+) and (P8+)FeII(H2O)(NO+), undergo subsequent reductive nitrosylation reactions that were found to be catalyzed by nitrite, which was also produced during the reaction. The effect of the nitrite concentration, pH, temperature, and pressure on the nitrite-catalyzed reductive nitrosylation process was studied in detail for (P8-)FeIII(H2O)2, (P8+)FeIII(H2O)2, and (P8+)FeIII(OH)(H2O), from which rate and activation parameters were obtained. On the basis of these data, we propose mechanistic pathways for the studied reactions. The available results favor the operation of an innersphere electron-transfer process between nitrite and coordinated NO(+). By way of comparison, the cationic porphyrin complex (P8+)FeIII(L)2 (L = H2O or OH-) was found to react with NO2(-) to yield the nitrite adduct (P8+)FeIII(L)(NO2)(-)). A detailed kinetic studied revealed that nitrite binds to (P8+)FeIII(H2O)2 according to a dissociative mechanism, whereas nitrite binding to (P8+)FeIII(OH)(H2O) at higher pH follows an associative mechanism, similar to that reported for the binding of NO to these complexes.     

Influence of an extremely negatively charged porphyrin on the reversible binding kinetics of NO to Fe(III) and the subsequent reductive nitrosylation

The polyanionic, water-soluble, and non-micro-oxo dimer-forming iron porphyrin (hexadecasodium iron 54,104,154,204-tetra-t-butyl-52,56,102,106,152,156,202,206-octakis[2,2-bis(carboxylato)ethyl]-5,10,15,20-tetraphenylporphyrin), (P16-)FeIII, with 16 negatively charged meso substituents on the porphyrin was synthesized and fully characterized by UV-vis and 1H NMR spectroscopy. A single pKa1 value of 9.90 +/- 0.01 was determined for the deprotonation of coordinated water in the six-coordinate (P16-)FeIII(H2O)2 and as attributed to the formation of the five-coordinate monohydroxo-ligated form, (P16-)FeIII(OH). The porphyrin complex reversibly binds NO in aqueous solution to yield the nitric oxide adduct, (P16-)FeII(NO+)(L), where L = H2O or OH-. The kinetics for the reversible binding of NO were studied as a function of pH, temperature, and pressure using the stopped-flow technique. The data for the binding of NO to the diaqua complex are consistent with the operation of a dissociative mechanism on the basis of the significantly positive values of DeltaS and DeltaV, whereas the monohydroxo complex favors an associatively activated mechanism as determined from the corresponding negative activation parameters. The rate constant, kon = 3.1 x 104 M-1 s-1 at 25 degrees C, determined for the NO binding to (P16-)FeIII(OH) at higher pH, is significantly lower than the corresponding value measured for (P16-)FeIII(H2O)2 at lower pH, namely, kon = 11.3 x 105 M-1 s-1 at 25 degrees C. This decrease in the reactivity is analogous to that reported for other diaqua- and monohydroxo-ligated ferric porphyrin complexes, and is accounted for in terms of a mechanistic changeover observed for (P16-)FeIII(H2O)2 and (P16-)FeIII(OH). The formed nitrosyl complex, (P16-)FeII(NO+)(H2O), undergoes subsequent reductive nitrosylation to produce (P16-)FeII(NO), which is catalyzed by nitrite produced during the reaction. Concentration-, pH-, temperature-, and pressure-dependent kinetic data are reported for this reaction. Data for the reversible binding of NO and the subsequent reductive nitrosylation reaction are discussed in reference to that available for other iron(III) porphyrins in terms of the influence of the porphyrin periphery.     

Odd-numbered Fe(III) complexes: synthesis, molecular structure, reactivity, and magnetic properties

Three isostructural disklike heptanuclear FeIII compounds of the general formula [FeIII7(mu3-O)3(L)3(mu-O2CCMe3)6(eta1-O2CCMe3)3(H2O)3], where L represents a di- or triethanolamine moiety, display a three-blade propeller topology, with the central Fe atom representing the axle or axis of the propeller. This motif corresponds to the theoretical model of a frustrated Heisenberg star, which is one of the very few solvable models in the area of frustrated quantum-spin systems and can, furthermore, be converted to an octanuclear cage for the case where L is triethanolamine to give [FeIII8(mu4O)3(mu4-tea)(teaH)3(O2CCMe3)6(N3)3].1/2MeCN.1/2H2O or [FeIII8(mu4O)3(mu4-tea)(teaH)3(O2CCMe3)6(SCN)3].2MeCN when treated with excess NaN3 or NH4SCN, respectively. The core structure is formally derived from that of the heptanuclear compounds by the replacement of the three aqua ligands by an {Fe(tea)} moiety, so that the 3-fold axis of the propeller is now defined by two central FeIII atoms. Magnetic studies on two of the heptanulcear compounds established unequivocally S = 5/2 spin ground state for these complexes, consistent with overall antiferromagnetic interactions between the constituent FeIII ions.     

Electrochemistry of xanthine oxidase and its interaction with nitric oxide.

With the help of nanocrystalline TiO2, the direct electrochemistry of xanthine oxidase (XOD) was achieved and two pairs of redox waves were observed. The interaction between XOD and nitric oxide (NO) was also investigated. The experimental results reveal that NO can be reduced at a XOD-nano TiO2 film modified electrode. When the NO concentration was low, the reduced product, HNO, would inactivate the protein. However, when the NO concentration was high, HNO would continue to react with NO to form N2O2- and N3O3-, which would not inhibit XOD, and thus the amount of active protein did not decrease any further.     

New insight into selective catalytic reduction of nitrogen oxides by ammonia over H-form zeolites: a theoretical study

Density functional theory calculations were carried out to investigate the reaction mechanism of selective catalytic reduction of nitrogen oxides by ammonia in the presence of oxygen at the Br?nsted acid sites of H-form zeolites. The Br?nsted acid site of H-form zeolites was modeled by an aluminosilicate cluster containing five tetrahedral (Al, Si) atoms. A low-activation-energy pathway for the catalytic reduction of NO was proposed. It consists of two successive stages: first NH(2)NO is formed in gas phase, and then is decomposed into N(2) and H(2)O over H-form zeolites. In the first stage, the formation of NH(2)NO may occur via two routes: (1) NO is directly oxidized by O(2) to NO(2), and then NO(2) combines with NO to form N(2)O(3), which reacts with NH(3) to produce NH(2)NO; (2) when NO(2) exceeds NO in the content, NO(2) associates with itself to form N(2)O(4), and then N(2)O(4) reacts with NH(3) to produce NH(2)NO. The second stage was suggested to proceed with low activation energy via a series of synergic proton transfer steps catalyzed by H-form zeolites. The rate-determining step for the whole reduction of NO(x) is identified as the oxidation of NO to NO(2) with an activation barrier of 15.6 kcal mol(-1). This mechanism was found to account for many known experimental facts related to selective catalytic reduction of nitrogen oxides by ammonia over H-form zeolites.     

Oxygen atom transfer from nitrite mediated by Fe(III) porphyrins in aqueous solution

Aqueous solutions of the iron(III) porphyrin complex FeIII(TPPS) (1, TPPS = tetra(4-sulfonatophenyl)-porphyrinato) and nitrite ion react with various substrates S to generate the ferrous nitrosyl complex FeII(TPPS)(NO) (2) plus oxidized substrate. When S is a water-soluble sulfonated phosphine, the product is the resulting monoxide. When air is introduced to the product solutions, 2 is rapidly reoxidized to 1; however, even in the absence of air, there is a slow regeneration of the ferric species with concomitant production of nitrous oxide. Thus, in an anaerobic aqueous environment, FeIII(TPPS) catalyzes oxygen atom transfer from nitrite ion to substrates with the eventual formation of N2O.     

微量Pt和Pd对Fe_2O_3/ZrO_2催化剂结构与性能的影响

用ＸＲＤ、ＴＰＲ及固定床微反应器技术研究了微量Ｐｔ 和Ｐｄ 对Ｆｅ２Ｏ３／ＺｒＯ２ 催化剂结构及性能的影响。结果表明，活性组分Ｆｅ３ ＋ 与载体ＺｒＯ２ 之间的相互作用会阻止载体从无定形ＺｒＯ２ 到ｔ－ＺｒＯ２ 进而ｍ － ＺｒＯ２ 的转变，同时，促进Ｆｅ２Ｏ３ 在ＺｒＯ２ 表面的分散。微量Ｐｔ 或Ｐｄ 的加入对Ｆｅ２Ｏ３ 在ＺｒＯ２ 上的分散状态无明显影响，但使Ｆｅ２Ｏ３／ＺｒＯ２ 的还原性能发生明显变化，表层Ｆｅ２Ｏ３ 的还原性显著增强，导致Ｍ－ Ｆｅ２Ｏ３／ＺｒＯ２ 催化剂在ＮＯ＋ ＣＯ反应中有更高的催化活性。Ｎ２ 生成的选择性次序为Ｆｅ２Ｏ３／ＺｒＯ２ ＞ Ｐｄ － Ｆｅ２Ｏ３／ＺｒＯ２ ＞ Ｐｔ－ Ｆｅ２Ｏ３／ＺｒＯ２ ＞ Ｐｄ／ＺｒＯ２ ＞ Ｐｔ／ＺｒＯ２ 。     

An iron-peroxo porphyrin complex: new synthesis and reactivity toward a Cu(II) complex giving a heme-peroxo-copper adduct

Side-on eta2-peroxo-iron porphyrins are strong nucleophiles. In cytochrome P450-like aromatase and other enzymes, such species are postulated as the active oxidants. In cytochrome c oxidase, hemea3-peroxo, hemea3-hydroperoxo, or hemea3-(mu-peroxo)-copper species are proposed as transient intermediates forming prior to O-O bond cleavage. In this report, we describe (1) a facile method for reduction of a heme-O2 species [(F8TPP)FeIII(O2-)(S)] (2), generating the ferric peroxo porphyrin complex [(F8TPP)FeIII(O22-)]- (3) (UV-vis, THF: lambdamax = 435 (Soret), 540(sh), 561; EPR: g = 8.7, 4.2), and (2) that this can be subsequently reacted with a ligand-copper(II) complex, [CuII(TMPA)-(CH3CN)](ClO4)2 (4), affording a heme-peroxo-copper heterobinuclear compound, [(F8TPP)FeII(O22-)-CuII(TMPA)](ClO4) (5). Generation of [(F8TPP)FeIII(O22-)]- (3) using cobaltocene as a one-electron reductant was monitored by UV-vis, EPR, and 1H NMR spectroscopies. Reaction between 3 and 4 was followed by UV-vis spectroscopy, and the product 5 could be precipitated and characterized. Coordination by copper(II) in 5 makes possible further reduction of the mu-peroxo complex by cobaltocene yielding the mu-oxo analogue, [(F8TPP)FeIII(O2-)-CuII(TMPA)](ClO4) (6).     

Role of protons in superoxide reduction by a superoxide reductase analogue

Superoxide reduction by thiolate-ligated [FeII(SMe2N4(tren))]+ (1) involves two proton-dependent steps and a single peroxide intermediate, [FeIII(SMe2N4(tren))(OOH)]+ (2). An external proton donor is required, ruling out mechanisms involving H+ or H-atom abstraction from the ligand N-H. The initial protonation step affording 2 occurs with fairly basic proton donors (EtOH, MeOH, NH4+) in THF. More acidic proton donors are required to cleave the Fe-O(peroxide) bond in MeOH, and this occurs via a dissociative mechanism. Reaction rates are dependent on the pKa of the proton donor, and a common [FeIII(SMe2N4(tren))(MeOH)]2+ (3) intermediate is involved. Acetic acid releases H2O2 from 2 under pseudo-first-order conditions ([HOAc] = 138 mM, [2] = 0.49 mM) with a rate constant of 8.2 x 10(-4) s(-1) at -78 degrees C in MeOH. Reduction of 3 with Cp2Co regenerates the active catalyst 1.     

In Process Citation

Knowing the values of the equilibrium constants corresponding to the reactions N(2)O(4) right harpoon over left harpoon 2NO(2) and N(2)O(4) right harpoon over left harpoon NO(+) + NO(3)(-) in sulpholane, we have undertaken the electrochemical study of N(2)O(4) by means of linear and cyclic voltammetry at the platinum electrode. The N(2)O(4) species undergoes one oxidation step N(2)O(4) right harpoon over left harpoon 2NO(2) right harpoon over left harpoon 2NO(2)(+) + 2e and two reduction steps NO(2) + N(2)O(4) + e(-)right harpoon over left harpoon N(2)O(3) + NO(3)(-) (1st wave), followed by 3N(2)O(4) + 2e(-) right harpoon over left harpoon 2N(2)O(3) + 2NO(3)(-), N(2)O(4) + e(-) right harpoon over left harpoon NO + NO(3)(-), 2N(2)O(3) + e(-) right harpoon over left harpoon 3NO + NO(3)(-) (2nd wave). The redox properties of N(2)O(4) are complicated by trace quantities of water because of the formation of the electroactive species N(2)O(3), HNO(3) and HNO(2) according to N(2)O(4) + H(2)O right harpoon over left harpoon HNO(2) + HNO(3) and N(2)O(4) + HNO(2) right harpoon over left harpoon N(2)O(3) + HNO(3). The standard potentials of the couples concerned have been evaluated and are discussed. sont discutés et évalués.     

Density functional theory modeling of the proposed nitrite anhydrase function of hemoglobin in hypoxia sensing

The role of NO and nitrite-bound methemoglobin (Hb(III)NO(2)(-)) in hypoxic signaling is highly controversial. One provoking possibility is that hemoglobin (Hb) functions as a nitrite anhydrase, producing N(2)O(3) (from nitrite) as an NO carrier. The ability of Hb to generate N(2)O(3) would provide an intriguing means of NO release from red blood cells. We have investigated this proposed new reactivity of Hb using density functional theory (DFT) calculations. For this purpose, models of the Hb/myoglobin (Mb) active site have been constructed. Our results show that the O-bound (nitrito) form of Hb/Mb(III)NO(2)(-) is essential for the formation of N(2)O(3). The formation and release of N(2)O(3) is shown to be energetically favorable by 1-3 kcal/mol, indicating that the anhydrase function of Hb/Mb is biologically feasible.     

Low-temperature reduction of NO(2) on oxidized Mo(110)

The reactions of nitrogen dioxide (NO(2)) were investigated on oxidized Mo(110) containing both chemisorbed oxygen and a thin film oxide. NO(2) reacts on both oxidized Mo(110) surfaces via a combination of reversible adsorption and reduction to NO, N(2), and trace amounts of N(2)O below 200 K. On the surface containing chemisorbed O, there is some complete dissociation of NO(2) to yield N(a) and O(a). N(2) forms at high temperatures through atom combination. On both surfaces, NO is the predominant product of NO(2) reduction. However, the chemisorbed layer which has a low oxidation state, and hence a greater capacity to accept oxygen, more effectively reduces NO(2). The selectivity for N(2) formation over N(2)O is greater for NO(2) as compared with NO on both surfaces studied. The selectivity changes are largely attributed to an increase in the concentration of Mo=O species and a change in the distribution of oxygen on the surface. Notably, more oxygen, in particular Mo=O moieties, is deposited by NO(2) reaction than by O(2) reaction, indicating that NO(2) is a stronger oxidant. The fact that there are several N-containing species on the surface at low temperatures may also affect the product distribution. On both surfaces, N(2)O(4), NO(2), and NO are identified by infrared spectroscopy upon adsorption at 100 K. All N(2)O(4) desorbs by 200 K, leaving only NO(2) and NO on the surface. Infrared spectroscopy of NO(2) on (18)O-labeled surfaces provides evidence for oxygen transfer or exchange between different types of sites even at low temperatures.     

Theoretical reduction potentials for nitrogen oxides from CBS-QB3 energetics and (C)PCM solvation calculations

The complete basis set method, CBS-QB3, is used in combination with two continuum solvation models for aqueous solvation to compute reduction potentials previously determined experimentally for 36 nitrogen oxides and related species of the general formula H(V)C(W)N(X)O(Y)Cl(Z). The PCM model led to the correlation E(o)exp (vs NHE) = 0.84E(o)calc + 0.03 V with an average error of 0.12 V (2.8 kcal/mol) and a maximum error of 0.32 V (7.4 kcal/mol). The CPCM/UAKS model gave E(o)exp (vs NHE) = 0.83E(o)calc + 0.11 V with the same average error. This general method was used to predict reduction potentials (+/-0.3 V) for nitrogen oxides for which reduction potentials are not known with certainty: NO2/NO2- (0.6 V), NO3/NO3- (1.9 V), N2O3-/N2O3(2-) (0.5 V), HN2O3/HN2O3- (0.9 V), HONNO,H+/HONNOH (1.6 V), 2NO,H+/HONNO (0.0 V), 2NO/ONNO- (-0.1 V), ONNO-/ONNO(2-) (-0.4 V), HNO,H+/H2NO (0.6 V), H2NO,H+/H2NOH (0.9 V), HNO,2H+/H2NOH (0.8 V), and HNO/HNO- (-0.7 V).     

Nitric oxide adsorption and reduction reaction mechanism on the Rh7(+) cluster: a density functional theory study

The transition metal rhodium has been proved the effective catalyst to convert from NO(x) to N(2.) In the present work, we are mainly focused on the NO adsorption and decomposition reaction mechanism on the surface of the Rh(7)(+) cluster, and the calculated results suggest that the reaction can proceed via three steps. First, the NO can adsorb on the surface of the Rh(7)(+) cluster; second, the NO decomposes to N and O atoms; finally, the N atom reacts with the second adsorbed NO and reduces to a N(2) molecule. The N-O bond breaks to yield N and O atoms in the second step, which is the rate-limiting step of the whole catalytic cycle. This step goes over a relatively high barrier (TS(12)) of 39.6 kcal/mol and is strongly driven by a large exothermicity of 55.1 kcal/mol during the formation of stable compound 3, accompanied by the N and O atoms dispersed on the different Rh atoms of the Rh(7)(+) cluster. In addition, the last step is very complex due to the different possibilities of reaction mechanism. On the basis of the calculations, in contrast to the reaction path II that generates N(2) from two nitrogen atoms coupling, the reaction path I for the formation of intermediate N(2)O is found to be energetically more favorable. Present work would provide some valuable fundamental insights into the behavior of the nitric oxide adsorption and reduction reaction mechanism on the Rh(7)(+) cluster.     

Catalytic two-electron reductions of N2O and N3- by myoglobin in surfactant films

Myoglobin (Mb), in films of dimethyldidodecylammonium bromide (ddab) on graphite electrodes, is used as a catalyst to mediate the electrochemical reduction of nitrous oxide (N2O) as well as the isoelectronic ion azide (N3-) in aqueous solutions. The electrocatalytic reductions are characterized by a rate-dependent irreversibility in cyclic voltammograms of Mb/ddab in the presence of the substrates. Bulk electrolysis shows that the reduction of 15N15NO by Mb/ddab yields 15N15N as shown by GC/MS. The catalytic reduction of azide results in almost quantitative formation of ammonia. These electrocatalytic processes are rationalized as two-electron reductions, with the catalyst cycling between the Fe(I) and Fe(III) states of Mb. To our knowledge, this is the first characterization of N2O reduction by an Fe porphyrin or heme protein.     

Theoretical Study of NO Dimer Adsorption and Dissociation on the CuCr2O4 （100） Surface

Theoretical simulation of the adsorption and dissociation of two NO molecules at the Cu^2＋, Cr^3＋ and bridge Cr^3＋ sites （b-Cr^3＋） on the normal spinel CuCr2O4 （100） surface has been carried out by density functional theory calculations. The results show that the formed N-down and O-down NO dimers are negatively charged. The formation of stable O-down dimers on the surface leads to the great elongation of N-O bond, which contributes to the NO reduction. The transition-state calculations indicate that the decomposition of O-down NO dimer at the b-Cr^3＋ site is most favorable and N2O is the major reduction product.     

Kinetic and spectroscopic studies of N694C lipoxygenase: a probe of the substrate activation mechanism of a nonheme ferric enzyme

Lipoxygenases (LOs) comprise a class of substrate activating mononuclear nonheme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. A commonly proposed mechanism for LO catalysis involves H-atom abstraction by an FeIII-OH- site, best described as a proton coupled electron transfer (PCET) process, followed by direct reaction of O2 with the resulting substrate radical to yield product. An alternative mechanism that has also been discussed involves the abstraction of a proton from the substrate by the FeIII-OH leading to a sigma-organoiron intermediate, where the subsequent sigma bond insertion of dioxygen into the C-Fe bond completes the reaction. H-atom abstraction is favored by a high E(o) of the FeII/FeIII couple and high pK(a) of water bound to the ferrous state, while an organoiron mechanism would be favored by a low E(o) (to keep the site oxidized) and a high pK(a) of water bound to the ferric state (to deprotonate the substrate). A first coordination sphere mutant of soybean LO (N694C) has been prepared and characterized by near-infrared circular dichroism (CD) and variable-temperature, variable-field (VTVH) magnetic circular dichroism (MCD) spectroscopies (FeII site), as well as UV/vis absorption, UV/vis CD, and electron paramagnetic resonance (EPR) spectroscopies (FeIII site). These studies suggest that N694C has a lowered E degrees of the FeII/FeIII couple and a raised pKa of water bound to the ferric site relative to wild type soybean lipoxygenase-1 (WT sLO-1) which would favor the organoiron mechanism. However, the observation in N694C of a significant deuterium isotope effect, anaerobic reduction of iron by substrate, and a substantial decrease in k(cat) (approximately 3000-fold) support H-atom abstraction as the relevant substrate-activation mechanism in sLO-1.     

Sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations on superoxide reductase: role of the axial thiolate in reactivity

Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN- bound low-spin FeIII forms of the 1Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin FeIII-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the FeIII bond to the proximal oxygen of this hydroperoxo species, which raises its pKa by an additional 5 log units relative to the pKa of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin FeIII-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C FeIII product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an FeIV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C FeIII site that results from the product release at the end of the O2- reduction cycle is calculated to be capable of reacting with a second O2-, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the 5C FeIII site of SOR, which is more positively charged, is calculated to have a high affinity for binding a sixth anionic ligand, which would inhibit its SOD activity.     

Distinguishing sources of N2O in European grasslands by stable isotope analysis

Nitrifiers and denitrifiers are the main producers of the greenhouse gas nitrous oxide (N(2)O). Knowledge of the respective contributions of each of these microbial groups to N(2)O production is a prerequisite for the development of effective mitigation strategies for N(2)O. Often, the differentiation is made by the use of inhibitors. Measurements of the natural abundance of the stable isotopes of N and O in N(2)O have been suggested as an alternative for the often unreliable inhibition studies. Here, we tested the natural abundance incubation method developed by Tilsner et al.1 with soils from four European grasslands differing in long-term management practices. Emission rates of N(2)O and stable isotope natural abundance of N(2)O and mineral N were measured in four different soil incubations: a control with 60% water-filled pore space (WFPS), a treatment with 60% WFPS and added ammonium (NH(4) (+)) to support nitrifiers, a control with 80% WFPS and a treatment with 80% WFPS and added nitrate (NO(3) (-)) to support denitrifiers. Decreases in NH(4) (+) concentrations, linked with relative (15)N-enrichment of residual NH(4) (+) and production of (15)N-depleted NO(3) (-), showed that nitrification was the main process for mineral N conversions. The N(2)O production, however, was generally dominated by reduction processes, as indicated by the up to 20 times larger N(2)O production under conditions favouring denitrification than under conditions favouring nitrification. Interestingly, the N(2)O concentration in the incubation atmospheres often levelled off or even decreased, accompanied by increases in delta(15)N and delta(18)O values of N(2)O. This points to uptake and further reduction of N(2)O to N(2), even under conditions with small concentrations of N(2)O in the atmosphere. The measurements of the natural abundances of (15)N and (18)O proved to be a valuable integral part of the natural abundance incubation method. Without these measurements, nitrification would not have been identified as essential for mineral N conversions and N(2)O consumption could not have been detected.