Nitrite has been shown in the past decade to be an important source of nitric oxide that acts as a vasodilator and intrinsic signaling molecule. [1] Numerous studies have proved that nitrite can be reduced in vivo, either non-enzymatically [2a,b] or enzymatically, in reactions catalyzed by xanthine oxidase, [2c] deoxyhaemoglobin, [2d] deoxymyoglobin, [2e] cytochrome c, [2f] or by thiol and metal-center-assisted processes inside the cell. [2g] The mechanism of the last process has recently been studied in detail, and has demonstrated that thiols (cysteine and gluthathione) stimulate water-soluble Fe 3+ -porphyrins to have nitrite reductase activity through an oxygen atom transfer (OAT) mechanism (Scheme 1) that leads to increased formation of Fe 2+ -NO-porphyrin and the corresponding sulfenic acid. [3a] Furthermore, the same research group showed recently that in fact the main products of this reaction are NO and its reduced congener, nitroxyl (HNO). [3b] However, the smallest thiol that can diffuse without any constrains [4a] and which could reach the active sites of most of the metalloproteins, that is, hydrogen sulfide, has not yet been considered as a possible regulator of this reaction. Nonetheless, in clinical praxis, nitrite is used as an antidote for Scheme 1. OAT mechanism. [3]

Readily exchangeable water molecules are commonly found in the active sites of oxidoreductases, yet the overwhelming majority of studies on small-molecule mimics of these enzymes entirely ignores the contribution of water to the reactivity. Studies of how these enzymes can continue to function in spite of the presence of highly oxidizing species are likewise limited. The mononuclear Mn complex with the potentially hexadentate ligand N-(2-hydroxy-5-methylbenzyl)-N,N',N'-tris(2-pyridinylmethyl)-1,2-ethanediamine (L) was previously found to act as both a HO-responsive MRI contrast agent and a mimic of superoxide dismutase (SOD). Here, we studied this complex in aqueous solutions at different pH values in order to determine its (i) acid-base equilibria, (ii) coordination equilibria, (iii) substitution lability and operative mechanisms for water exchange, (iv) redox behavior and ability to participate in proton-coupled electron transfer (PCET) reactions, (v) SOD activity and reductive activity toward both oxygen and superoxide, and (vi) mechanism for its transformation into the binuclear Mn complex with L-L and its hydroxylated derivatives. The conclusions drawn from potentiometric titrations, low-temperature mass spectrometry, temperature- and pressure-dependent O NMR spectroscopy, electrochemistry, stopped-flow kinetic analyses, and EPR measurements were supported by the structural characterization and quantum chemical analysis of proposed intermediate species. These comprehensive studies enabled us to determine how transiently bound water molecules impact the rate and mechanism of SOD catalysis. Metal-bound water molecules facilitate the PCET necessary for outer-sphere SOD activity. The absence of the water ligand, conversely, enables the inner-sphere reduction of both superoxide and dioxygen. The L complex maintains its SOD activity in the presence of OH and Mn-oxo species by channeling these oxidants toward the synthesis of a functionally equivalent binuclear Mn species.

While nitric oxide (NO, nitrogen monoxide) is a critically important signaling agent, its cellular concentrations must be tightly controlled, generally through its oxidative conversion to nitrite (NO2−) where it is held in reserve to be reconverted as needed. In part, this reaction is mediated by the binuclear heme a3/CuB active site of cytochrome c oxidase. In this report, the oxidation of NO(g) to nitrite is shown to occur efficiently in new synthetic µ-oxo heme-FeIII−O−CuII(L) constructs (L being a tridentate or tetradentate pyridyl/alkylamino ligand), and spectroscopic and kinetic investigations provide detailed mechanistic insights. Two new X-ray structures of µ-oxo complexes have been determined and compared to literature analogs. All µ-oxo complexes react with 2 mol equiv NO(g) to give 1:1 mixtures of discrete [(L)CuII(NO2−)]+ plus ferrous heme-nitrosyl compounds; when the first NO(g) equiv reduces the heme center and itself is oxidized to nitrite, the second equiv of NO(g) traps the ferrous heme thus formed. For one µ-oxo heme-FeIII−O−CuII(L) compound, the reaction with NO(g) reveals an intermediate species (“intermediate”), formally a bis-NO adduct, [(NO)(porphyrinate)FeII-(NO2−)−CuII(L)]+ (λmax = 433 nm), confirmed by cryo-spray ionization mass spectrometry and EPR spectroscopy, along with the observation that cooling a 1:1 mixture of [(L)CuII(NO2−)]+ and heme-FeII(NO) to −125 °C leads to association and generation of the key 433 nm UV–vis feature. Kinetic-thermodynamic parameters obtained from low-temperature stopped-flow measurements are in excellent agreement with DFT calculations carried out which describe the sequential addition of NO(g) to the µ-oxo complex.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Nitrite has been shown in the past decade to be an important source of nitric oxide that acts as a vasodilator and intrinsic signaling molecule. [1] Numerous studies have proved that nitrite can be reduced in vivo, either non-enzymatically [2a,b] or enzymatically, in reactions catalyzed by xanthine oxidase, [2c] deoxyhaemoglobin, [2d] deoxymyoglobin, [2e] cytochrome c, [2f] or by thiol and metal-center-assisted processes inside the cell. [2g] The mechanism of the last process has recently been studied in detail, and has demonstrated that thiols (cysteine and gluthathione) stimulate water-soluble Fe 3+ -porphyrins to have nitrite reductase activity through an oxygen atom transfer (OAT) mechanism (Scheme 1) that leads to increased formation of Fe 2+ -NO-porphyrin and the corresponding sulfenic acid. [3a] Furthermore, the same research group showed recently that in fact the main products of this reaction are NO and its reduced congener, nitroxyl (HNO). [3b] However, the smallest thiol that can diffuse without any constrains [4a] and which could reach the active sites of most of the metalloproteins, that is, hydrogen sulfide, has not yet been considered as a possible regulator of this reaction. Nonetheless, in clinical praxis, nitrite is used as an antidote for Scheme 1. OAT mechanism. [3]