线粒体呼吸链的电子顺磁共振

Mitochondria respiratory chain studied by electron paramagnetic resonance spectroscopy

有氧呼吸(aerobic respiration)是地球上高等生物繁衍生息的生理基础,而使这个生理活动得以正常维持的基础是绿色植物的光合作用(photosynthesis)[1,2].后者利用太阳光能将CO2和H2O合成前者所需的富含化学能的有机物,同时释放出氧气以维持大气层中约21%的含氧量[1].巴随着人类社会的发展,人们生活水平日渐提高,人们也迫切希望了解人体自身的各种生命活动,以获得健康体质,达到延年益寿的目的.众所周知,人体各种生命活动所需的能量主要由有氧呼吸提供.因此,作为呼吸作用主体的线粒体,具有非常重要且不可替代的生理功能.比如,它一方面为其他生理活动提供能量,另一方面还维持这些生理活动的正常进行[2].当线粒体发生功能性障碍或生理活动失调时,会诱发许多疾病,甚至危及生命[3,4].

Aerobic respiration is the indispensable physiology of higher organism living on earth, since the required energy for biological activities is supplied mainly by this metabolism. It is well established that mitochondria plays an important role in converting the stable chemical energy stored in foods into the active and utilizable energy supplying to life activities. Upon the process, the energy conversion is carried out by the respiratory electron transport chain, which consists of five distinct and coupled protein complexes(termed complex Ⅰ-Ⅴ) embedded in the mitochondria intima. Among them, complex Ⅰ(NADH-ubiquinone oxidoreductase), Ⅲ(cytochrome bc1) and Ⅳ(cytochrome c oxidase) are the electron-coupled proton-pumping enzymes. Except for the complex Ⅴ(i.e., ATP synthase), each complex has several redox cofactors to promote the electron transfer within themselves, and the transfer from complex Ⅰ and Ⅱ to complex Ⅲ is shuttled by membrane-embedded ubiquinones, from complex Ⅲ to Ⅳ by the soluble cytochrome c. When the reductive electron is donated by the initial donors NADH or FADH2 to the final acceptor molecular oxygen O2, a transmembrane proton motive force is formed simultaneously. The latter is the driving force for the ATP synthesis via ATP synthase with a ratio of three protons per ATP. All the aforementioned processes are also known as oxidative phosphorylation, and the arrangement of the electron carriers therein is called as respiratory electron transport chain. In the stepwise electron relay, these carriers undergo the redox changes, giving rise to the respective paramagnetic and diamagnetic spin states. These single-electron relays are prone to the electron paramagnetic resonance spectroscopy(EPR), which is a powerful and noninvasive technique to monitor the unpaired electron in situ. As shown in the overwhelming literatures, EPR technique has been adopted to reveal the electron transfer pathway in different biological activities. Herein, the application of EPR to study the dynamic electron transfer in mitochondria is reviewed briefly. However, this conventional application required rather high concentration of the active center at a level of 1-10μmol/L. Actually, most of the intermediates of the electron carriers are very active and short-life. In the human body, only a few tissues can accumulate such required concentration of paramagnetic substances, e.g., in blood, heart and liver. For the past decade, a more sensitive single-molecule magnetic resonance technique based on the nitrogen-vacancy(NV) center of diamond has been developing dramatically. In principle, this advanced EPR technique requires the less sample or concentration, even down to the single biomolecule, and provides the time-resolved dynamic information. Lately, the studies of single-biomolecule EPR at room temperature or in aqueous solution have been reported consecutively in single protein(MAD2/mitotic arrest deficient-2) and single DNA(tethered DNA duplexes). Finally, a perspective of single-biomolecule magnetic resonance technique is given for the further study on mitochondrion and relative physiological activities. The single molecule EPR technique is also helpful to unveil the other specific information of biological processes, for example, drug screening, personalized medicine and other major applications.