53-84-9Relevant articles and documents
Oxygen Activation and Electron Transfer in Flavocytochrome P450 BM3
Ost, Tobias W. B.,Clark, Jonathan,Mowat, Christopher G.,Miles, Caroline S.,Walkinshaw, Malcolm D.,Reid, Graeme A.,Chapman, Stephen K.,Daff, Simon
, p. 15010 - 15020 (2003)
In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s-1. The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.
The Peroxidase-NADH Biochemical Oscillator. 1. Examination of Oxygen Mass Transport, the Effect of Light, and the Role of Methylene Blue
Olson, Dean L.,Scheeline, Alexander
, p. 1204 - 1211 (1995)
The peroxidase-NADH oscillator examined here initially consists of four chemical components.The well-mixed aqueous solution includes native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.In this system, the macroscopic appearance of the process of oxygen dissolution from the gas phase is dependent on k-m, the mass transport constant of oxygen out of solution.Additional details of oxygen mass transport are derived.The amplitude of oxygen oscillations is decreased by continuous illumination by the deuterium source of a diode array spectrophotometer.This attenuation effect of light is dependent on wavelengths =+ allows several damped oscillations of small amplitude.Subsequent addition of MB+ to the oscillator results in oscillations of much larger amplitude.MB+ is seen to either directly or indirectly enhance the conversion of peroxidase compound III to the native enzyme and then inhibit oxygen consumption, allowing the initiation of relatively large, prolonged oscillations.MB+ is seen to function either as a system catalyst, or as a peroxidase inhibitor in the oxidation of NADH by oxygen.
NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role
Cortial, Sylvie,Chaignon, Philippe,Iorga, Bogdan I.,Aymerich, Stephane,Truan, Gilles,Gueguen-Chaignon, Virginie,Meyer, Philippe,Morera, Solange,Ouazzani, Jamal
, p. 3916 - 3922 (2010)
NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD+ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H2O2 produced during the oxidative process or added exogenously. Structured summary: MINT- 7990140: nfrA1 (uniprotkb:. P39605) and nfrA1 (uniprotkb:. P39605) bind (MI:. 0407) by X-ray crystallography (MI:. 0114).
The Peroxidase-NADH Biochemical Oscillator. 2. Examination of the Roles of Hydrogen Peroxide and Superoxide
Olson, Dean L.,Scheeline, Alexander
, p. 1212 - 1217 (1995)
The peroxidase-NADH oscillator examined here initially consists of a well-mixed aqueous solution of native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.Hydrogen peroxide and superoxide have been implicated as important chemical intermediates.A comprehensive model which includes such intermediates and all initial chemical species has appeared elsewhere.To experimentally explore the role of hydrogen peroxide in the oscillator, H2O2 was substituted for MB+ as an initial ingredient.This substitution allows relatively small, quasi-sinusoidal oscillations sensitive to the oxygen mass transport constant, and predicted earlier in a theoretical model.The oscillations become much larger when MB+ is added, suggesting that MB+ might serve as a chemical mediator between the small oscillations seen when H2O2 is substituted for MB+, and the relatively large oscillations observed when MB+ is present.Catalase and superoxide dismutase are used as enzymatic scavengers for H2O2 and O2.-, respectively.The enzymes are added individually to a working oscillator at oxygen minima and maxima to examine the roles and approximate the concentrations of H2O2 and O2.-.For the enzyme addition experiments, a perturbation model for oxygen behavior is proposed and applied to the interpretation of experimental data.Two methods of analysis for the addition of the enzyme probes indicate a higher concentration of H2O2 and O2.- at oxygen maxima than at minima.Comparison of experimental and simulated data indicate that the relatively simple model presented here is a resonable, yet apparently incomplete, representation of oxygen dynamics for the addition of scavenger enzymes to this oscillator.
Magneto-stimulated hydrodynamic control of electrocatalytic and bioelectrocatalytic processes.
Katz, Eugenii,Willner, Itamar
, p. 10290 - 10291 (2002)
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Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis
Khan, Md. Iqbal Hassan,Kim, Hyeung,Ashida, Hiroyuki,Ishikawa, Takahiro,Shibata, Hitoshi,Sawa, Yoshihiro
, p. 1802 - 1805 (2005)
The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with k cat values 3.45 and 5.68s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.
Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features
Feldmann, Jonas,Li, Yao,Tor, Yitzhak
, p. 4379 - 4389 (2019/03/07)
Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD+) has emerged as a crucial substrate for NAD+-consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD+ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD+ surrogate, termed NthAD+, are described. Its preparation was accomplished by enzymatic conversion of synthetic thATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD+ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, NthAD+ was reduced by alcohol dehydrogenase (ADH) to NthADH and afforded thADP-ribose (thADPr) upon hydrolysis by NAD+-nucleosidase (NADase). Furthermore, NthAD+ was engaged in cholera toxin A (CTA)-catalyzed mono(thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(thADP-ribosyl)ation. Due to its high photophysical responsiveness, NthAD+ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD+ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD+) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD+ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.
Organometallic ruthenium and iridium phosphorus complexes: Synthesis, cellular imaging, organelle targeting and anticancer applications
Li, JuanJuan,Tian, Zhenzhen,Zhang, Shumiao,Xu, Zhishan,Mao, Xudong,Zhou, Yumin,Liu, Zhe
, (2019/02/20)
The use of metal complexes containing phosphorus ligands as anticancer agents has not been well studied. In this work, eight novel half-sandwich IrIII and RuII compounds with P^P-chelating ligands have been synthesized and fully characterized, and alongside two crystal structures were reported. All eight complexes displayed highly potent antiproliferative activity, up to nine times more potent than the clinical anticancer drug cisplatin towards A549 lung cancer cells. Complex Ir1, which has a simpler structure and highly potent antiproliferative activity, was selected to investigate in further mechanistic studies. No hydrolysis and nucleobase binding occurred for complex Ir1. In order to elucidate subcellular localization, the self-luminescence of the complex Ir1 was utilized. Ir1 can specifically target lysosomes and facilitate excessive production of reactive oxygen species, resulting in lysosomal membrane permeabilization in A549 cells. Release of cathepsin B and changes in the mitochondria membrane potential also contributed to the observed cytotoxicity of Ir1, which demonstrated an anticancer action mechanism that was different from that of cisplatin. The favorable results from biological and chemical research demonstrated that these types of complexes hold significant theranostic potential.