Krishna M C, Grahame D A, Samuni A, Mitchell J B, Russo A
Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5537-41. doi: 10.1073/pnas.89.12.5537.
Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, the mechanism of superoxide (.O2-) dismutation by various five-membered ring and six-membered ring nitroxides was studied by electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, and bulk electrolysis. Electron paramagnetic resonance signals from the carbocyclic nitroxide derivatives (piperidinyl, pyrrolidinyl, and pyrrolinyl) were unchanged when exposed to enzymatically generated .O2-, whereas, in the presence of .O2- and reducing agents such as NADH and NADPH, the nitroxides underwent reduction to their respective hydroxylamines. The reaction of 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (Tempol-H) with .O2- was measured and, in agreement with earlier reports on related compounds, the rate was found to be too slow to be consistent with a mechanism of .O2- dismutation involving the hydroxylamine as an intermediate. Voltammetric analyses of the carbocyclic nitroxide derivatives revealed a reversible one-electron redox couple at positive potentials. In contrast, oxazolidine derivatives were irreversibly oxidized. At negative potentials, all of the nitroxides studied exhibited a broad, irreversible reductive wave. The rate of .O2- dismutation correlated with the reversible midpoint redox potential. Bulk electrolysis at positive potentials was found to generate a metastable oxidized form of the nitroxide. The results indicate that the dismutation of .O2- is catalyzed by the oxoammonium/nitroxide redox couple for carbocyclic nitroxide derivatives. In addition to the one-electron mitochondrial reduction pathway, the present results suggest the possibility that cellular bioreduction by a two-electron pathway may occur subsequent to oxidation of stable nitroxides. Furthermore, the cellular destruction of persistent spin adduct nitroxides might also be facilitated by a primary univalent oxidation.
超氧化物的歧化反应此前已被证明可由稳定的氮氧化物化合物催化。在本研究中，通过电子顺磁共振光谱法、紫外可见分光光度法、循环伏安法和本体电解法研究了各种五元环和六元环氮氧化物对超氧化物（·O₂⁻）的歧化机制。当碳环氮氧化物衍生物（哌啶基、吡咯烷基和吡咯啉基）暴露于酶促产生的·O₂⁻时，其电子顺磁共振信号未发生变化，然而，在·O₂⁻和诸如NADH和NADPH等还原剂存在的情况下，氮氧化物会被还原为各自的羟胺。对4 - 羟基 - 2,2,6,6 - 四甲基 - 1 - 羟基哌啶（Tempol - H）与·O₂⁻的反应进行了测定，与先前关于相关化合物的报道一致，发现该反应速率过慢，不符合以羟胺为中间体的·O₂⁻歧化机制。对碳环氮氧化物衍生物的伏安分析表明，在正电位下存在一个可逆的单电子氧化还原对。相比之下，恶唑烷衍生物被不可逆地氧化。在负电位下，所有研究的氮氧化物均表现出一个宽的、不可逆的还原波。·O₂⁻歧化反应的速率与可逆中点氧化还原电位相关。发现在正电位下进行本体电解会产生氮氧化物的亚稳氧化形式。结果表明，对于碳环氮氧化物衍生物，·O₂⁻的歧化反应是由氧鎓铵/氮氧化物氧化还原对催化的。除了单电子线粒体还原途径外，本研究结果还表明，稳定氮氧化物氧化后，可能会通过双电子途径发生细胞生物还原。此外，持久性自旋加合物氮氧化物的细胞破坏也可能通过一次单价氧化而得到促进。
