Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.     

Ferryl and hydroxy intermediates in the reaction of oxygen with reduced cytochrome c oxidase

Cytochrome c oxidase catalyses the 4-electron reduction of dioxygen to water and translocates protons vectorially across the inner mitochondrial membrane. Proposed reaction pathways for the catalytic cycle of the O2 reduction are difficult to verify without knowing the structures of the intermediates, but we now have such information for the catalytic intermediates in the first steps of the reaction of O2 with cytochrome c oxidase from resonance Raman spectroscopy, a technique that enables iron-ligand stretching modes to be identified. Here we report on two more key intermediates: a ferryl-oxo (Fe4 = O2-) and a ferric-hydroxy (Fe3+--OH-) intermediate at the level of 3- and 4-electron reduction, respectively. We identified these intermediates by their characteristic iron-oxygen stretching frequencies (786 cm-1 for Fe4+ = O2-, and 450 cm-1 for Fe3+ -- OH-) and oxygen and deuterium isotope shifts. The oxo atom in the ferryl intermediate is hydrogen-bonded and the iron-oxygen bond in the hydroxy intermediate is anomalously weak. With the identification of the primary, ferryl and hydroxy intermediates, the predominant structures at almost all stages of O2 reduction are now known and the catalytic pathway can be described with more certainty.     

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.     

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.     

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.     

Structure of cytochrome c nitrite reductase.

The enzyme cytochrome c nitrite reductase catalyses the six-electron reduction of nitrite to ammonia as one of the key steps in the biological nitrogen cycle, where it participates in the anaerobic energy metabolism of dissimilatory nitrate ammonification. Here we report on the crystal structure of this enzyme from the microorganism Sulfurospirillum deleyianum, which we solved by multiwavelength anomalous dispersion methods. We propose a reaction scheme for the transformation of nitrite based on structural and spectroscopic information. Cytochrome c nitrite reductase is a functional dimer, with 10 close-packed haem groups of type c and an unusual lysine-coordinated high-spin haem at the active site. By comparing the haem arrangement of this nitrite reductase with that of other multihaem cytochromes, we have been able to identify a family of proteins in which the orientation of haem groups is conserved whereas structure and function are not.     

Coherent dynamics of a flux qubit coupled to a harmonic oscillator

In the emerging field of quantum computation and quantum information, superconducting devices are promising candidates for the implementation of solid-state quantum bits (qubits). Single-qubit operations, direct coupling between two qubits and the realization of a quantum gate have been reported. However, complex manipulation of entangled states-such as the coupling of a two-level system to a quantum harmonic oscillator, as demonstrated in ion/atom-trap experiments and cavity quantum electrodynamics-has yet to be achieved for superconducting devices. Here we demonstrate entanglement between a superconducting flux qubit (a two-level system) and a superconducting quantum interference device (SQUID). The latter provides the measurement system for detecting the quantum states; it is also an effective inductance that, in parallel with an external shunt capacitance, acts as a harmonic oscillator. We achieve generation and control of the entangled state by performing microwave spectroscopy and detecting the resultant Rabi oscillations of the coupled system.     

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.     

纳米火棉胶膜自组装细胞色素c的直接电化学研究

用纳米火棉胶膜将细胞色素c固定在玻碳电极表面,制备了细胞色素c-火棉胶膜修饰电极.吸附在火棉胶膜上的细胞色素c可以与电极发生直接电子传递.在pH=7.0的0.1mol/LPBS缓冲溶液中可得到一对准可逆的细胞色素c的血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对氧化还原峰,实验求得细胞色素c异相电子传递速率常数k0为65.4μm/s.进一步考察了扫速、溶液pH值等因素对细胞色素c电子传递的影响,并用电化学阻抗法研究了修饰电极的电化学行为.     

The a.c. and d.c. Josephson effects in a Bose-Einstein condensate

The alternating- and direct-current (a.c. and d.c.) Josephson effects were first discovered in a system of two superconductors, the macroscopic wavefunctions of which are weakly coupled via a tunnelling barrier. In the a.c. Josephson effect, a constant chemical potential difference (voltage) is applied, which causes an oscillating current to flow through the barrier. Because the frequency is proportional to the chemical potential difference only, the a.c. Josephson effect serves as a voltage standard. In the d.c. Josephson effect, a small constant current is applied, resulting in a constant supercurrent flowing through the barrier. In a sense, the particles do not 'feel' the presence of the tall tunnelling barrier, and flow freely through it with no driving potential. Bose-Einstein condensates should also support Josephson effects; however, while plasma oscillations have been seen in a single Bose-Einstein condensate Josephson junction, the a.c. Josephson effect remains elusive. Here we observe the a.c. and d.c. Josephson effects in a single Bose-Einstein condensate Josephson junction. The d.c. Josephson effect has been observed previously only in superconducting systems; in our study, it is evident when we measure the chemical potential-current relation of the Bose-Einstein condensate Josephson junction. Our system constitutes a trapped-atom interferometer with continuous readout, which operates on the basis of the a.c. Josephson effect. In addition, the measured chemical potential-current relation shows that the device is suitable for use as an analogue of the superconducting quantum interference device, which would sense rotation.     

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.     

Effect of impulse blockage on cytochrome oxidase activity in monkey visual system

Cytochrome oxidase (cytochrome c oxidase; ferrocytochrome c: oxygen oxidoreductase, EC 1.9.2.1) has been introduced as an oxidative metabolic marker for neurones in the central nervous system. Previous studies have shown that mature neurones remained sensitive to altered functional demands, and that both developing and adult neurones responded to sensory deprivation or deafferentation by reducing their cytochrome oxidase (Cyt. Ox.) activity. More recently, we showed that the blockage of retinal impulse transmission with tetrodotoxin led to a reversible reduction in Cyt. Ox. staining of affected lateral geniculate (LGN) and striate neurones in adult cats. The present study sought to extend these findings to adult monkeys, where Cyt. Ox. 'puffs' or 'blobs' are uniquely present in the visual cortex. We found that, while the retina remained histologically intact, with only moderate decreases in Cyt. Ox. staining of large ganglion cells and the two plexiform layers, subtle changes occurred in the LGN as early as 1 day post-tetrodotoxin injection, and clear reduction in enzyme levels was evident in both the LGN and the visual cortex by 3 days. Changes became progressively more severe up to 4 weeks post-injection. Within area 17, alternating bands of high and low Cyt. Ox. staining occurred in lamina IV, with alternating rows of dark and lightly reactive puffs superimposed in exact register. Thus, the mature visual neurones in the primate remain extremely sensitive to the cessation of retinal impulse transmission, and plastic metabolic changes occur through several synapses along the sensory pathway.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Identification of the electron transfers in cytochrome oxidase that are coupled to proton-pumping

Mitochondrial cytochrome oxidase is a functionally complex, membrane-bound respiratory enzyme which catalyses both the reduction of O2 to water and proton-pumping. During respiration, an exogenous donor, cytochrome c, donates four electrons to O2 bound at the bimetallic haem alpha 3 Fe-Cu centre within the enzyme. These four electron transfers are mediated by the enzyme's haem alpha and CuA redox centres and result in the translocation of four protons across the inner mitochondrial membrane. The molecular mechanism of proton translocation has not yet been delineated, however, and in the absence of direct experimental evidence all four electron transfers have been assumed to couple equally to proton-pumping. Here, I report the effects of proton-motive force and membrane potential on two equilibria involving intermediates of the bimetallic centre at different levels of O2 reduction. The results show that only two of the electron transfers, to the 'peroxy' and 'oxyferryl' intermediates of the bimetallic centre, are linked to proton translocation, a finding which strongly constrains candidate mechanisms for proton-pumping.