Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step.
Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step.
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DOI：
10.3390/molecules23071672
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发表时间：
2018-07-09
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影响因子：
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通讯作者：
Čėnas N
中科院分区：
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作者：
Valiauga B;Misevičienė L;Rich MH;Ackerley DF;Šarlauskas J;Čėnas N
氧不敏感的NAD（P）H：硝基还原酶（NR）通过亚硝基（Ar-NO）中间体将硝基芳烃（Ar-NO2）还原为羟胺（Ar-NHOH）。还原型烟酰胺腺嘌呤二核苷酸或其磷酸盐NAD（P）H可直接或酶促还原Ar-NO，但哪一过程在NR的催化中占主导地位尚不清楚。我们发现E.大肠杆菌NR-A（NfsA）对2 mol NADPH/mol TNT和4 mol NADPH/mol Tetryl的氧化能力分别为100%和100%。抗坏血酸的加入使Ar-NO还原为Ar-NHOH，使NADPH/Ar-NO2的化学计量比变为1：1（TNT）和2：1（tetryl），并降低NADPH氧化的速率。抗坏血酸盐不干扰NfsA还原醌类过程中NADPH的氧化。我们的抗坏血酸抑制模式和酶和非酶还原亚硝基苯的分析表明，直接还原Ar-NO的NADPH，而不是酶还原是硝基芳香还原过程中的主要机制。
Oxygen-insensitive NAD(P)H:nitroreductases (NR) reduce nitroaromatics (Ar-NO2) into hydroxylamines (Ar-NHOH) through nitroso (Ar-NO) intermediates. Ar-NO may be reduced both enzymatically and directly by reduced nicotinamide adenine dinucleotide or its phosphate NAD(P)H, however, it is unclear which process is predominant in catalysis of NRs. We found that E. coli NR-A (NfsA) oxidizes 2 mol of NADPH per mol of 2,4,6-trinitrotoluene (TNT) and 4 mol of NADPH per mol of tetryl. Addition of ascorbate, which reduces Ar-NO into Ar-NHOH, changes the stoichiometry NADPH/Ar-NO2 into 1:1 (TNT) and 2:1 (tetryl), and decreases the rate of NADPH oxidation. Ascorbate does not interfere with the oxidation of NADPH during reduction of quinones by NfsA. Our analysis of ascorbate inhibition patterns and both enzymatic and non-enzymatic reduction of nitrosobenzene suggests that direct reduction of Ar-NO by NADPH rather than enzymatic reduction is the predominant mechanism during nitroaromatic reduction.