Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tC^O), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

The kinetics of the reaction of hydroxyl radical (OH) with trimethyl phosphate (CH₃O)₃PO (TMP) (reaction (1)) OH + TMP → products (1) was studied at the bath gas (He) pressure of 1 bar over the 273–837 K temperature range. Hydroxyl radicals were produced in fast reactions of electronically excited oxygen atoms O(¹D) with either H₂O or H₂. Excited oxygen atoms O(¹D) were produced by photolysis of ozone, O₃, at 266 nm (4^th harmonic of Nd:YAG laser) over the 273–470 K temperature range and by photolysis of N₂O at 193 nm (ArF excimer laser) over the whole temperature range including the elevated temperature range 470–837 K. The reaction rate constant exhibits a V-shaped temperature dependence, negative in the low temperature range, 273–470 K (the rate constant decreases with temperature), and positive in the elevated temperature range, 470–837 K (the rate constant increases with temperature), with a turning point at 471 K. The rate constant could be fairly well fitted with the three parameter modified Arrhenius expression, k₁ = 7.52 × 10⁻¹⁸ (T/298)⁹ exp(34 367 J mol⁻¹/RT) cm³ per molecule per s (273–837 K). Previously, only one indirect experimental measurement at a single (ambient) temperature was available. The temperature dependence over an extended temperature range obtained in this study together with the peculiar V-shaped temperature dependence will have an impact on the modelling of the flame inhibition by phosphates as well on the further understanding of the mechanisms of elementary chemical reactions.

The kinetics of the reaction of hydroxyl radical (OH) with dimethyl methylphosphonate (DMMP, (CH₃O)₂CH₃PO) (reaction 1) OH + DMMP → products (1) was studied at the bath gas (He) pressure of 1 bar over the 295–837 K temperature range. Hydroxyl radicals were produced in the fast reaction of electronically excited oxygen atoms O(¹D) with H₂O. The time-resolved kinetic profiles of hydroxyl radicals were recorded via UV absorption at around 308 nm using a DC discharge H₂O/Ar lamp. The reaction rate constant exhibits a pronounced V-shaped temperature dependence, negative in the low temperature range, 295–530 K (the rate constant decreases with temperature), and positive in the elevated temperature range, 530–837 K (the rate constant increases with temperature), with a turning point at 530 ± 10 K. The rate constant could not be adequately fitted with a standard 3-parameter modified Arrhenius expression. The data were fitted with a 5-parameter expression as: k₁ = 2.19 × 10⁻¹⁴(T/298)^2.43exp(15.02 kJ mol⁻¹/RT) + 1.71 × 10⁻¹⁰exp(−26.51 kJ mol⁻¹/RT) cm³molecule⁻¹s⁻¹ (295–837 K). In addition, a theoretically predicted pressure dependence for such reactions was experimentally observed for the first time.