Single-electron Reduction Potential Research Articles

A universal molecular oxygen-mediated photocatalysis strategy to boost visible-light induced hydrogen evolution through partial water splitting

A universal oxygen-mediated, stepwise strategy is proposed for efficiently inducing visible-light photocatalytic partial water decomposition into hydrogen over various semiconductor photocatalysts with conduction band bottoms below the single-electron oxygen reduction potential. In this scenario, molecular O2 can be transformed into reactive oxygen species, serving as both an oxidant and a homogeneous catalyst for producing hydrogen from alkaline aqueous solution containing various organic substrates. Further enhancement the performance is achieved by doping with phosphorous and oxygen, which constructs a local internal electric field and introduces sulfur vacancies, thereby facilitating the transport of photogenerated charge carriers, particularly on a representative CdS photocatalyst. The optimal hydrogen evolution performance reaches 2321.4 and 8521.4 μmol·gcatatlyst−1·h−1 in methanol and formaldehyde solution systems, respectively, with an apparent quantum efficiency exceeding 59.4 % under 450 nm visible light irradiation. Mechanistic studies demonstrate that the oxygen-mediated, sequential single-electron transfer process can occur with virtually zero activation energy.

The Catalysis Mechanism of E. coli Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies.

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be -215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π-π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents.

Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells.

NADPH:thioredoxin reductase (TrxR) is considered a potential target for anticancer agents. Several nitroheterocyclic sulfones, such as Stattic and Tri-1, irreversibly inhibit TrxR, which presumably accounts for their antitumor activity. However, it is necessary to distinguish the roles of enzymatic redox cycling, an inherent property of nitroaromatics (ArNO2), and the inhibition of TrxR in their cytotoxicity. In this study, we calculated the previously unavailable values of single-electron reduction potentials of known inhibitors of TrxR (Stattic, Tri-1, and 1-chloro-2,4-dinitrobenzene (CDNB)) and inhibitors identified (nitrofuran NSC697923 and nitrobenzene BTB06584). These calculations were according to the rates of their enzymatic single-electron reduction (PMID: 34098820). This enabled us to compare their cytotoxicity with that of model redox cycling ArNO2. In MH22a and HCT-116 cells, Tri-1, Stattic, CDNB, and NSC697023 possessed at least 10-fold greater cytotoxicity than can be expected from their redox cycling activity. This may be related to TrxR inhibition. The absence of enhanced cytotoxicity in BTB06548 may be attributed to its instability. Another known inhibitor of TrxR, tetryl, also did not possess enhanced cytotoxicity, probably because of its detoxification by DT-diaphorase (NQO1). Apart from the reactions with NQO1, the additional mechanisms influencing the cytotoxicity of the examined inhibitors of TrxR are their reactions with cytochromes P-450. Furthermore, some inhibitors, such as Stattic and NSC697923, may also inhibit glutathione reductase. We suggest that these data may be instrumental in the search for TrxR inhibitors with enhanced cytotoxic/anticancer activity.

Thermodynamics Evaluation of Selective Hydride Reduction for α,β-Unsaturated Carbonyl Compounds

The selective reduction of α,β-unsaturated carbonyl compounds is one of the core reactions and also a difficult task for organic synthesis. We have been attempting to study the thermodynamic data of these compounds to create a theoretical basis for organic synthesis and computational chemistry. By electrochemical measurement method and titration calorimetry, in acetonitrile at 298 K, the hydride affinity of two types of unsaturated bonds in α,β-unsaturated carbonyl compounds, their single-electron reduction potential, and the single-electron reduction potential of the corresponding radical intermediate are determined. Their hydrogen atom affinity, along with the hydrogen atom affinity and proton affinity of the corresponding radical anion, is also derived separately based on thermodynamic cycles. The above data are used to establish the corresponding "Molecule ID Card" (Molecule identity card) and analyze the reduction mechanism of unsaturated carbonyl compounds. Primarily, the mixture of any carbonyl hydride ions and Ac-tempo+ will stimulate hydride transfer process and create corresponding α,β-unsaturated carbonyl compounds and Ac-tempoH from a thermodynamic point of view.

Modified quantum mechanical approach for the estimation of single-electron reduction potential of nitroaromatic compounds in aqueous medium

The midpoint single-electron reduction potential of nitroaromatic compounds in aqueous medium at pH 7.0 (potential of ArNO2/ArNO2 · – couple, E m7) frequently determines their therapeutic and/or toxic properties. However, its estimation remains a complex problem. We propose a modified method of E m7 estimation by quantum mechanical calculations, based on the use of the dielectric continuum model together with a certain number of H2O molecules at the vicinity of nitro group. The optimal number of H2O molecules corresponds to a minimal difference between the experimentally determined and calculated values of E m7, and/or the most negative value of calculated E m7. This enabled us to calculate the E m7 values for a number of ArNO2 (n = 19) with the average deviation of 0.027 V from the experimentally determined ones. Apart from nitrobenzene derivatives, the application of this approach for the representatives of nitropyridines, nitrofurans, nitrothiophenes, and nitrothiazoles was demonstrated. In this case, nitroimidazole derivatives are an exception, evidently due to a strong proton accepting properties of N3 atom of their free radicals.

The comparative study of redox properties of recombinant human cytosolic and mitochondrial NADPH: thioredoxin reductases

We compared the redox properties of recombinant cytosolic (TrxR1) and mitochondrial (TrxR2) isoforms of human flavosulfoselenoenzyme NADPH:thioredoxin reductase. The standard redox potentials of isoenzymes (E0 7), determined according to the redox equilibrium with the NADP+/NADPH couple, were equal to –0.295 V (TrxR1) and –0.270 V (TrxR2). The more positive value of E0 7 of TrxR2 may be attributed to the presence of His-125 at the vicinity of catalytic disulfide and selenylsulfide, instead of Tyr-116 in TrxR1. The reactivity of several quinones and nitroaromatic compounds towards TrxR1 and TrxR2 increased with their single-electron reduction potential (E1 7). For the first time, we studied the TrxR1-catalysed reduction of a series of aromatic N-oxides which were reduced in a mixed single- and two-electron way. Their reactivity was close to that of quinones and nitroaromatics with the similar values of E1 7.

QSARs in prooxidant mammalian cell cytotoxicity of nitroaromatic compounds: the roles of compound lipophilicity and cytochrome P-450- and DT-diaphorase-catalyzed reactions

Frequently, the aerobic mammalian cell cytotoxicity of nitroaromatic compounds (ArNO2) increases with their single-electron reduction potential (E17), thus reflecting the relationship between their enzymatic single-electron reduction rate and E17. This shows that the main factor of ArNO2 cytotoxicity is redox cycling and oxidative stress. In this work, we found that the reactivity of a series of nitrobenzenes, nitrofurans and nitrothiophenes towards single-electron transferring NADPH:cytochrome P-450 reductase and adrenodoxin reductase/adrenodoxin increases with their E17. However, their cytotoxicity in mouse hepatoma MH22a and human colon carcinoma HCT-116 cells exhibited a poorly expressed dependence on E17. The correlations were significantly improved after the introduction of compound octanol/water distribution coefficient at pH 7.0 (log D) as a second variable. This shows that the lipophilicity of ArNO2 enhances their cytotoxicity. The inhibitors of cytochromes P-450, α-naphthoflavone, isoniazid and miconazole, and an inhibitor of DT-diaphorase, dicoumarol, in most cases decreased the cytotoxicity of several randomly chosen compounds. This shows that the observed cytotoxicity vs E17 relationships in fact reflect the superposition of several cytotoxicity mechanisms.

A Simple Computational Tool for Accurate, Quantitative Prediction of One-Electron Reduction Potentials of Hypoxia-Activated Tirapazamine Analogues.

The reduction potentials of bioreductively-activated drugs represent an important design parameter to be accommodated in the course of creating lead compounds and improving the efficacy of older generation drugs. Reduction potentials are traditionally reported as single-electron reduction potentials, E(1), measured against reference electrodes under strictly defined experimental conditions. More recently, computational chemists have described redox properties in terms of a molecule's highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), in electron volts (eV). The relative accessibility of HOMO/LUMO data through calculation using today's computer infrastructure and simplified algorithms make the calculated value (LUMO) attractive in comparison to the accepted but rigorous experimental determination of E(1). This paper describes the correlations of eV (LUMO) to E(1) for three series of bioreductively-activated benzotriazine di-N-oxides (BTDOs), ring-substituted BTDOs, ring-added BTDOs and a selection of aromatic nitro compounds. The current computational approach is a closed-shell calculation with a single optimization. Gas phase geometry optimization was followed by a single-point DFT (Density Functional Theory) energy calculation in the gas phase or in the presence of polar solvent. The resulting DFT-derived LUMO energies (eV) calculated for BTDO analogues in gas phase and in presence of polar solvent (water) exhibited very strong linear correlations with high computational efficiency (r2 = 0.9925) and a very high predictive ability (MAD = 7 mV and RMSD = 9 mV) when compared to reported experimentally determined single-electron reduction potentials.

Charge trapping and transfer mechanisms of noble metals and metal oxides deposited Ga2O3 toward typical contaminant degradation

Ga2O3 highlights the high energy band structure for redox reaction. However, information on noble metals and metal oxides deposited Ga2O3 for contaminant elimination and the corresponding mechanisms is still lack. Here, metals and metal oxides deposited Ga2O3 (labeled as M-P and MO-C; M = Ag, Pt, and Pd) were synthesized by photocatalytic and chemical deposition methods and confirmed with XRD and XPS. M-P and MO-C were applied in photocatalytic degradation of three typical contaminants: ciprofloxacin, ronidazole and rhodamine B. The ciprofloxacin and ronidazole degradation rates by Ag2O-C and rhodamine B degradation rate by Ag-P were 3.0, 2.7 and 2.4 times as high as those by pure Ga2O3. This research demonstrates that photocatalytic activities of M-P and MO-C are affected by the types and oxidation states of noble metals, as well as the target contaminants. Potentials of M or MO (E(MΦ) or E(MOCB), defined as work function of the metals or conduction band of the metal oxides) is the determining factor for both charge trapping and charge transfer abilities. The highest photocatalytic activity can be obtained when the E(MΦ) or E(MOCB) was kept at the level just beyond the single electron reduction potential of O2, such as Ag-P and Ag2O-C. Finally, the contaminants-specific photocatalytic activities were related to the different functional reactive species. Findings of this study provide important information for the rational design of M and MO deposited photocatalysts.

Preparation and preliminary evaluation of a tris-metronidazole-99mTc(CO)3 complex for targeting tumor hypoxia

Conventional 99mTc-radiopharmaceuticals for the detection of tumor hypoxia generally possess a single nitroimidazole moiety. Herein, we report the synthesis and evaluation of a 99mTc-complex with three-nitroimidazole moieties in an attempt to improve hypoxic cell detection. Isocyanide derivative of metronidazole (MetroNC) was synthesized and subsequently radiolabeled with [99mTc(CO)3(H2O)3]+ precursor complex, wherein the three labile water molecules were replaced with MetroNC ligand to form a pseudo-octahedral [99mTc(CO)3(MetroNC)3]+ complex. Analysis of corresponding Re(CO)3-analog prepared in macroscopic scale confirmed the formation of expected complex. Cyclic voltammetric studies of [Re(CO)3(MetroNC)3]+ complex showed no significant change in single-electron reduction potential (SERP) of MetroNC ligand (− 0.96 V) upon forming the [Re(CO)3(MetroNC)3]+ complex (− 0.90 V). In vitro studies in Chinese hamster ovary (CHO) cells showed three-fold preferential accumulation of [99mTc(CO)3(MetroNC)3]+ complex in hypoxic cells over normoxic cells. Biodistribution studies of [99mTc(CO)3(MetroNC)3]+ complex in Swiss mice bearing fibrosarcoma tumor showed tumor uptake and steady retention till 60 min post injection. Present study constitutes a novel design approach towards development of a 99mTc-radiopharmaceutical for hypoxia imaging application, which could be extended to other potential nitroimidazole ligands.

Effective Artificial Co-enzyme Based on Single-Electron Reduced Form of 2,2′-Bipyridinium Salt Derivatives for Formate Dehydrogenase in the Catalytic Conversion of CO2 to Formic Acid

Abstract Formate dehydrogenase (FDH) is a useful biocatalyst for CO2 reduction to formic acid in a photoredox system consisting of a photosensitizer and an electron carrier. The electron carrier, single-electron reduced 2,2′-bipyridinium salts (2,2′-BP2+s) act as the co-enzyme for FDH in the reaction of CO2 to formic acid. An advantage of 2,2′-BP2+s is the easy change of structural geometry and the various single-electron reduction potentials. For further improvement of CO2 reduction catalytic activity of FDH, various 2,2′-BP2+s were synthesized as effective artificial co-enzymes for FDH. The effect of the structural geometry and the single-electron reduction potential in the single-electron reduced form of 2,2′-BP2+s on the CO2 reduction catalytic activity of FDH was studied by enzymatic kinetic analysis in detail for the first time. Especially, the catalytic efficiency, kcat/Km value of the single-electron reduced 1,1′-ethylene-2,2′-bipyridinium salt was c.a. 126 times larger than that of native co-enzyme, NADH. These results showed that catalytic activity of FDH can be manipulated with complete control by using 2,2′-BP2+ without changing the structure of FDH and has opened a new avenue for the approach of NAD+/NADH redox free system with FDH using an inexpensive small electron carrier molecule.

Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin

In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe3+, metHb) convert NO. into nitrate at the expense of NADH and O2. FHbs contribute to bacterial resistance to nitrosative stress. Therefore, inhibition of FHbs functions may decrease the pathogen virulence. We report here a kinetic study of the reduction of quinones and nitroaromatic compounds by S. aureus FHb. We show that this enzyme rapidly reduces quinones and nitroaromatic compounds in a mixed single- and two-electron pathway. The reactivity of nitroaromatics increased upon an increase in their single-electron reduction potential (E17), whereas the reactivity of quinones poorly depended on their E17 with a strong preference for a 2-hydroxy-1,4-naphthoquinone structure. The reaction followed a ‘ping-pong’ mechanism. In general, the maximal reaction rates were found lower than the maximal presteady-state rate of FAD reduction by NADH and/or of oxyhemoglobin (HbFe2+O2) formation (~130 s−1, pH 7.0, 25 °C), indicating that the enzyme turnover is limited by the oxidative half-reaction. The turnover studies showed that quinones prefreqently accept electrons from reduced FAD, and not from HbFe2+O2. These results suggest that quinones and nitroaromatics act as ‘subversive substrates’ for FHb, and may enhance the cytotoxicity of NO. by formation of superoxide and by diverting the electron flux coming from reduced FAD. Because quinone reduction rate was increased by FHb inhibitors such as econazole, ketoconazole, and miconazole, their combined use may represent a novel chemotherapeutical approach.

Influence of platinum metals on spectral and electrochemical characteristics of metallated azole luminophors

Spectral (1H NMR and IR), optical, and voltammetry parameters of Pd(II), Pt(II), Rh(III), and Ir(III) complexes with ethylenediamine and metallated azole luminophors [2-phenylbenzoxazole, 2-phenylbenzothiazole, 2,5-diphenyloxazole, 2-(1,1'-biphenyl)-4-yl]-5-phenyloxazole, and 2-(naphthalene-l-yl)benzothiazole] have been studied. The variation in spectroscopic parameters of the complexes is related to the increase in the efficiency of donor-acceptor interaction of Pt(II) and Ir(III) with the ligands as compared to Pd(II) and Rh(III). The correlation between the energy of the metal-ligand charge transfer bands and the difference between the single-electron oxidation and reduction potentials of the complexes is observed. Quenching of the fluorescence of metallated azoles and phosphorescence of the complexes in the visible region from the metal-modified intraligand excited state are related with the increase in the efficiency of the singlettriplet conversion due to the spin-orbital interaction in platinum metals.

Reduction of quinones and nitroaromatic compounds by Escherichia coli nitroreductase A (NfsA): Characterization of kinetics and substrate specificity

NfsA, a major FMN-associated nitroreductase of E. coli, reduces nitroaromatic compounds via consecutive two-electron transfers. NfsA has potential applications in the biodegradation of nitroaromatic environment pollutants, e.g. explosives, and is also of interest for the anticancer strategy gene-directed enzyme prodrug therapy. However, the catalytic mechanism of NfsA is poorly characterized. Here we examined the NADPH-dependent reduction of quinones (n = 16) and nitroaromatic compounds (n = 12) by NfsA. We confirmed a general “ping-pong” reaction scheme, and preliminary rapid reaction studies of the enzyme reduction by NADPH showed that this step is much faster than the steady-state turnover number, i.e., the enzyme turnover is limited by the oxidative half-reaction. The reactivity of nitroaromatic compounds (log kcat/Km) followed a linear dependence on their single-electron reduction potential (E17), indicating a limited role for compound structure or active site flexibility in their reactivity. The reactivity of quinones was lower than that of nitroaromatics having similar E17 values, except for the significantly enhanced reactivity of 2-OH-1,4-naphthoquinones, consistent with observations previously made for the group B nitroreductase of Enterobacter cloacae. We present evidence that the reduction of quinones by NfsA is most consistent with a single-step (H−) hydride transfer mechanism.

Quinone- and nitroreductase reactions of Thermotoga maritima thioredoxin reductase.

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E(1)7, -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E(1)7, thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31±0.03 V at pH 7.0, which may correspond to the FAD/FADH2 redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role.

A study on nitroimidazole-99mTc(CO)3 complexes as hypoxia marker: some observations towards possible improvement in in vivo efficacy.

Hypoxia plays a negative role in the clinical management of cancer. Detection of hypoxic status of a cancer is important for selecting patients for hypoxia directed therapy. Though [(18)F]fluoromisonidazole ([(18)F]FMISO), a PET radiopharmaceutical, is presently being used in the clinic for the detection of hypoxia, considering the logistical advantages of (99m)Tc and wider availability of SPECT scanners, a radiopharmaceutical based on this isotope may find wider applicability. Nine nitroimidazole (2-, 4- and 5-nitroimidazole) ligands were synthesized and radiolabeled using [(99m)Tc(CO)3(H2O)3](+) precursor to obtain a group of complexes possessing different single electron reduction potential (SERP), overall charge and lipophilicity, the three attributes which decide the efficacy of the complex to detect hypoxic cells in vivo. The nitroimidazole-(99m)Tc(CO)3 complexes as well as [(18)F]FMISO were evaluated in fibrosarcoma tumor bearing mice. The (99m)Tc(CO)3 complexes of nitroimidazole iminodiacetic acid (IDA) showed better tumor uptake and retention than nitroimidazole diethylenetriamine (DETA) and nitroimidazole aminoethylglycine (AEG) complexes. Tumor uptake observed with [(18)F]FMISO was higher than any of the nitroimidazole-IDA- (99m)Tc(CO)3 complexes. However, [(18)F]FMISO clearance from tumor was found to be faster compared to 2-nitroimidazole-IDA-(99m)Tc(CO)3 complex. Observed tumor uptake and retention of the radiotracers evaluated could be correlated to its blood clearance pattern and SERP. Results of the present study indicated that uptake of the radiotracer in tumor is closely associated with its rate of clearance from blood. The study also indicated that along with SERP, clearance of radiotracer from blood (net effect of charge and lipophilicity) is a critical factor which decides the in vivo efficacy of the hypoxia detecting radiopharmaceutical.

Redox properties and prooxidant cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX).

In order to characterize the possible mechanism(s) of cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX) we examined the redox properties of DNQX, and its mononitro- (NQX) and denitro- (QX) derivatives. The irreversible electrochemical reduction of the nitro groups of DNQX was characterized by the reduction peak potentials (Ep,7) of -0.43 V and -0.72 V vs. Ag/AgCl at pH 7.0, whereas NQX was reduced at Ep,7 = -0.67 V. The reactivities of DNQX and NQX towards the single-electron transferring enzymes NADPH:cytochrome P-450 reductase and NADPH:adrenodoxin reductase/adrenodoxin complex were similar to those of model nitrobenzenes with the single-electron reduction potential (E¹₇) values of -0.29 V - -0.42 V. DNQX and NQX also acted as substrates for two-electron transferring mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase). The cytotoxicity of DNQX in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was prevented by antioxidants and an inhibitor of NQO1, dicoumarol, and was enhanced by the prooxidant alkylating agent 1,3-bis(2-chloromethyl)-1-nitrosourea. A comparison with model nitrobenzene compounds shows that the cytotoxicity of DNQX and NQX reasonably agrees with the ease of their electrochemical reduction, and/or their reactivities towards the used enzymatic single-electron reducing systems. Thus, our data imply that the cytotoxicity of DNQX in FLK cells is exerted mainly through oxidative stress.

Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase.

We examined the kinetics of single-electron reduction of a large number of structurally diverse quinones and nitroaromatic compounds, including a number of antitumour and antiparasitic drugs, and nitroaromatic explosives by recombinant rat neuronal nitric oxide synthase (nNOS, EC 1.14.13.39), aiming to characterize the role of nNOS in the oxidative stress-type cytotoxicity of the above compounds. The steady-state second-order rate constants (kcat/Km) of reduction of the quinones and nitroaromatics varied from 10² M⁻¹s⁻¹ to 10⁶ M⁻¹s⁻¹, and increased with an increase in their single-electron reduction potentials (E¹₇). The presence of Ca²⁺/calmodulin enhanced the reactivity of nNOS. These reactions were consistent with an 'outer sphere' electron-transfer mechanism, considering the FMNH∙/FMNH₂ couple of nNOS as the most reactive reduced enzyme form. An analysis of the reactions of nNOS within the 'outer sphere' electron-transfer mechanism gave the approximate values of the distance of electron transfer, 0.39-0.47 nm, which are consistent with the crystal structure of the reductase domain of nNOS. On the other hand, at low oxygen concentrations ([O₂] = 40-50 μM), nNOS performs a net two-electron reduction of quinones and nitroaromatics. This implies that NOS may in part be responsible for the bioreductive alkylation by two-electron reduced forms of antitumour aziridinyl-substituted quinones under a modest hypoxia.

Preparation and evaluation of a 99mTcN–PNP complex of sanazole analogue for detecting tumor hypoxia

A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to that of misonidazole, was synthesized and radiolabeled with [99mTcN(PNP)] precursor to evaluate its potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good yields and challenging studies with cysteine showed stability of the complex against trans-chelation. However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insufficient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present 99mTc-complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios. However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor, limit the utility of the complex to detect tumor hypoxia.

Quinone- and nitroreductase reactions of Thermotoga maritima peroxiredoxin–nitroreductase hybrid enzyme

Thermotoga maritima peroxiredoxin–nitroreductase hybrid enzyme (Prx–NR) consists of a FMN-containing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromatics by NR proceeded in a two-electron manner, and follows a ‘ping-pong’ scheme with sometimes pronounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx–NR was characterized by the linear dependence of their reactivity (logkcat/Km) on their single-electron reduction potentials E71, while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the specificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx–NR, and to a significant diversity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics.