A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

2-氨基-3-苯并恶唑喹啉水复合物激发态分子内与分子间质子转移的竞争机理

基于密度泛函与含时密度泛函理论方法, 研究2-氨基-3-苯并恶唑喹啉（ABO）水复合物(ABO-H₂O)激发态分子内与分子间质子转移的竞争机理. 结果表明： ABO-H₂O复合物中存在一个分子内的氢键和两个分子间的氢键; 基态ABO-H₂O复合物被激发至第一电子激发态后, 仅需越过一个34.157 kJ/mol能垒, 复合物即可发生激发态分子内的质子转移反应; ABO-H₂O复合物激发态分子间的双质子转移过程中存在一个63.585 kJ/mol能垒; 在第一电子激发态上, ABO-H₂O复合物中存在分子内与分子间质子转移的竞争机制; ABO-H₂O复合物更易出现激发态分子内的质子转移过程.     

Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.     

铜离子影响腺嘌呤内及其碱基对间质子转移的理论研究

在B3LYP／6.31+G*水平研究了结合在腺嘌呤N7位的铜离子对腺嘌呤内及AT、AU碱基对间质子转移反应的影响。气相中铜离子有利于腺嘌呤内的质子转移,其中Cu2+的作用比Cu+的更明显。单水合铜离子的静电作用被水部分屏蔽,这不利于腺嘌呤分子内的质子转移反应。当Cu+与腺嘌呤N7位结合,同时水分子协助质子转移时,腺嘌呤内的质子转移反应将很容易发生。另一方面,本研究按单质子分步转移对铜离子影响碱基对间质子转移设计了两条路径:腺嘌呤先质子化再去质子化(Path1)和腺嘌呤先去质子化再质子化(Path2)。分析各构型的相对能发现:① 对Cu2+-AT(或Cu2+-AU)体系,Cu2+可以稳定碱基对间单质子转移后形成的离子碱基对构型,而且Cu2+导致各碱基酸性增加,有利于发生单质子转移;② 对Cu2+-AT体系,单质子转移反应的主路径为Path2,然而该反应生成的离子碱基对可以很容易地生成Cu2+-AT,所以Cu2+-AT更趋向于不发生质子转移,与［AT］+体系相比,Cu2+-AT体系也不发生双质子转移;③ 对Cu2+-AU体系,单双质子转移的反应均能发生,但双质子转移的反应比单质子转移的困难。与［AU］+体系相比,单质子转移反应中两条路径存在竞争,双质子转移路径更趋向于Path2。     

Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

7-羟基黄酮分子在水中分子内电荷转移及分子间多重质子的转移

基于密度泛函和含时密度泛函理论的B3LYP方法, 在TZVP基组水平上模拟7-羟基黄酮(7HF)的水复合物--7HF-(H₂O)4激发态的动力学机制. 结果表明： 复合物中7HF在第一电子激发态的前线分子轨道出现分子内电荷转移过程; 复合物中7HF在基态和第一电子激发态发生了分子结构扭转, 复合物中5个分子间的氢键在第一电子激发态均出现加强机制, 该机制可以驱动激发态的多重质子转移反应.     

Reversible redox energy coupling in electron transfer chains

Reversibility is a common theme in respiratory and photosynthetic systems that couple electron transfer with a transmembrane proton gradient driving ATP production. This includes the intensely studied cytochrome bc1, which catalyses electron transfer between quinone and cytochrome c. To understand how efficient reversible energy coupling works, here we have progressively inactivated individual cofactors comprising cytochrome bc1. We have resolved millisecond reversibility in all electron-tunnelling steps and coupled proton exchanges, including charge-separating hydroquinone-quinone catalysis at the Q(o) site, which shows that redox equilibria are relevant on a catalytic timescale. Such rapid reversibility renders popular models based on a semiquinone in Q(o) site catalysis prone to short-circuit failure. Two mechanisms allow reversible function and safely relegate short-circuits to long-distance electron tunnelling on a timescale of seconds: conformational gating of semiquinone for both forward and reverse electron transfer, or concerted two-electron quinone redox chemistry that avoids the semiquinone intermediate altogether.     

由4-氰基苄溴引发的苯乙烯电子活化再生原子转移自由基聚合

以4-氰基苄溴(CBB)为引发剂、Fe Cl3·6H2O/PPh3络合体系为催化剂、VC为还原剂实现了对苯乙烯(St)的电子活化再生原子转移自由基聚合(AGET ATRP)。研究表明,聚合过程中单体转化随反应时间增加而线性增长、数均分子量随单体转化率提高而线性增长、得到的聚合物分子量分布指数(PDI)在1.07~1.31之间。为进一步研究取代基团对聚合的影响,选用了4-甲基苄溴(MBB)为引发剂作了对比研究,所得聚合物的结构用核磁氢谱进行了验证。     

Directional electron transfer in ruthenium-modified horse heart cytochrome c

Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru-cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.     

Reduction of cytochrome c oxidase by a second electron leads to proton translocation

Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.     

N-(2-羟基苯亚甲基)苯胺衍生物分子内质子 转移取代基效应的密度泛函理论研究

在B3LYP/6-31+G(d,p)水平上研究了N-(2-羟基苯亚甲基)苯胺衍生物分子内质子转移的取代基效应.结果表明:吸电子基引入后会让分子更加趋于平面构型,而供电子基取代后让分子发生了一定程度的扭转;吸电子取代基引入后减小了分子平面的电子密度,拉近了N_1-H_2距离,增强了分子内氢键的强度,降低了醇式到酮式的结构互变能垒.供电子取代基引入后增加了分子平面的电子密度,加大了N_1-H_2间距,减弱了分子内氢键的强度,使得质子转移能垒升高.对分子的前沿轨道研究表明醇式较酮式结构来说更易形成激发态,并且能隙随着吸电子能力和供电子能力增强变小,说明其生物活性变强.     

氢键链中质子和电子的协同转移

提出氢键链中电荷转移的一种机制。电子被质子子晶格的反孤子缺陷所俘获。钟罩型电子波函数局域在质子子晶格的压缩区。氢键链中的质子和电子以电孤子－反孤子对束缚态的形式协同转移。     

Experimental measurement of proton conductivity and electronic conductivity of membrane electrode assembly for proton exchange membrane fuel cells

Charge transport processes involving the proton migration and electron transfer for different parts of membrane electrode assemble (MEA) play an essential role for developing the novel electrode and enhancing the electrochemical performance towards proton exchange membrane fuel cells (PEMFCs). However, the coupled charge transport processes make it difficult for evaluating proton conductivity and electronic conductivity of different parts in MEAs under operation conditions of fuel cells. Here in this work, we propose an experiment approach for separating the electronic conductivity and proton conductivity of different components of MEA at the operating conditions of PEMFCs. This approach involves two different measuring devices, which both consist of electron or proton conducting layers, sealing layers and sample layer, followed by tailoring the thickness of sample layers and via electrochemical impedance spectroscopy (EIS) to quantity the electronic conductivity and proton conductivity of different layers. These experiment results show the great potential in the development of different components of MEA.     

6-氯烟酰胺的异构化和质子迁移理论研究

在密度泛函理论B3LYP/6-31G(d)基组水平上,计算了气相中6-氯烟酰胺分子酮式和烯醇式质子迁移异构化过程的2种可能途径:分子内直接质子迁移和水分子辅助质子迁移.结果表明,在气相中只存在一种稳定构型,水分子的参与降低了质子迁移过程的活化能.     

2-巯基嘧啶互变异构的密度泛函理论计算

采用密度泛函理论,在B3LYP/6-3l1G**基组水平上,计算并考察了2-巯基嘧啶分子硫酮式和硫醇式结构进行结构互变的质子迁移过程中的2种可能途径:(a)分子内质子迁移,(b)水助质子迁移.计算结果表明,途经(b)所需的活化能较小,氢键在降低反应活化能方面起着重要作用.     

2-(2,5-二羟基苯基)-4(3H)-喹唑啉酮质子转移及七元瓜环限制作用的理论研究

在B3LYP/6-311++G(d)和ONIOM(B3LYP/6-311++g(d):PM3)水平上,研究了2-(2,5-二羟基苯基)-4(3H)-喹唑啉酮(DHPQ)及其在七元瓜环(CB7)限制下的双质子转移过程,确定了DHPQ分子各互变异构体的结构、能量以及基态质子转移反应的过渡态.通过对结构变化和能量差异的分析,探讨了DHPQ中的双质子转移机理,考察了质子转移的纳米笼效应以及该笼效应对质子转移热力学性质的影响,同时研究纳米腔与DHPQ的几何匹配关系以及水溶剂对包合物质子转移的影响.计算结果表明:DHPQ是采取分步转移机理,纳米腔的限制对质子转移过程产生一个很大的能垒,抑制分子内质子转移过程,水溶剂的引入促进Path 2,抑制Path 1.     

Structural Changes of Water Molecules Upon the Reduction of Quinones in The Reaction Center from Rhodobactery Sphaeroides

1 Results The photosynthetic bacterial reaction center (RC) is a membrane protein complex.The RC is composed of three protein subunits and redox components such as bacteriochlorophylls, bacteriopheophytins,and quinones.The RC performs the photochemical electron transfer from the bacteriochlorophyll dimer through a series of electron donor and acceptor molecules to a secondary quinone,QB.QB accepts electrons from a primary quinone,QA,in two sequential electron transfer reactions.The second electron trans...     

氧化锌纳米晶\卟啉复合材料能量及电子转移过程

利用溶胶-凝胶方法制备了半导体ZnO纳米材料,用混合方法将ZnO与卟啉有机物(对-二羟基苯基卟啉)形成复合体系。通过对复合体系光学性质变化的研究,建立了半导体晶体-有机化合物的能级结构图,提出了纳米材料———有机物复合体系中的能量及电子转移过程[1]。     

Structure dependence of intramolecular photoinduced electron transfer in 9-aminoacridine benzoyl esters dyads linked by a polyether chain

Conclusions Both experimental observations and theoretical calculations indicated that: (I) The flexible dyads reported in this paper exist preferentially in folded conformations in which the donor and the acceptor are held in close proximity. This allows overlap of the orbitals of each moiety leading to very efficient intramolecular fluorescence quenching. (ii) Quenching of the fluorescence of the donor in the dyads via electron transfer depends on the electron affinity of the acceptor. (iii) The electron-attracting ability of the acceptors in the dyads is strongly influenced by the type and relative spatial disposition of the substituents.