Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli -: Changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III)
Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli -: Changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III)
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DOI：
10.1074/jbc.m306251200
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发表时间：
2003-10-24
影响因子：
4.8
通讯作者：
Linn, S
中科院分区：
文献类型：
--
作者：
Brumaghim, JL;Li, Y;Linn, S
在体内，DNA会因由亚铁离子（Fe²⁺）和细胞还原剂（如NADH，它可将铁离子（Fe³⁺）还原为亚铁离子（Fe²⁺）并使铁循环利用）介导的芬顿反应而受损。为了研究大肠杆菌对这种循环的反应，在过氧化氢（H₂O₂）刺激后，对参与烟酰胺核苷酸代谢的几种酶的活性进行了测定。依赖NADPH的过氧化物酶、NADH/NADP⁺转氢酶和葡萄糖 - 6 - 磷酸脱氢酶被诱导的程度最强，增加了2.5 - 3倍。此外，在分别暴露于0.5 mM或5 mM H₂O₂后15分钟，细胞内NADPH与NADH的比率分别增加了6倍或92倍。在体外，尽管NADH和NADPH的还原电位相同，但NADH被Fe³⁺氧化的速度比NADPH快多达16倍。为了理解这种速率差异，通过¹H、¹³C和³¹P核磁共振波谱研究了Fe³⁺和Ga³⁺与NAD(P)H的相互作用。与NADH的结合主要发生在腺嘌呤的N7位和氨基上，但对于NADPH，在2'-磷酸基团处也有强烈的金属相互作用。M³⁺（Fe³⁺或Ga³⁺）与腺嘌呤环的相互作用会使其在NAD(P)H的折叠形式下与具有氧化还原活性的烟酰胺环紧密靠近，但M³⁺与2'-磷酸基团的相互作用会避免这种紧密接触。此外，通过吸收光谱测定，Fe³⁺·NADPH复合物的电荷转移态能量明显高于Fe³⁺·NADH复合物。因此我们认为，在暴露于H₂O₂时，NADH库会被耗尽，而与Fe³⁺反应性较弱的NADPH则作为主要的烟酰胺核苷酸还原剂发挥作用。
DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP(+) transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mM H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P) H were examined by H-1, C-13, and P-31 NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD( P) H, but interaction of M3+ with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+.NADPH complex than for the Fe3+.NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant.