E-cadherin-dependent stimulation of traction force at focal adhesions via the Src and PI3K signaling pathways.

The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (µFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on µFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior.

Interquinone electron transfer in photosystem I as evidenced by altering the hydrogen bond strength to the phylloquinone(s).

The kinetics of electron transfer from phyllosemiquinone (PhQ(*-)) to the iron sulfur cluster F(X) in Photosystem I (PS I) are described by lifetimes of approximately 20 and approximately 250 ns. These two rates are attributed to reactions involving the quinones bound primarily by the PsaB (PhQ(B)) and PsaA (PhQ(A)) subunits, respectively. The factors leading to a approximately 10-fold difference between the observed lifetimes are not yet clear. The peptide nitrogen of conserved residues PsaA-Leu722 and PsaB-Leu706 is involved in asymmetric hydrogen-bonding to PhQ(A) and PhQ(B), respectively. Upon mutation of these residues in PS I of the green alga, Chlamydomonas reinhardtii , we observe an acceleration of the oxidation kinetics of the PhQ(*-) interacting with the targeted residue: from approximately 255 to approximately 180 ns in PsaA-L722Y/T and from approximately 24 to approximately 10 ns in PsaB-L706Y. The acceleration of the kinetics in the mutants is consistent with a perturbation of the H-bond, destabilizing the PhQ(*-) state, and increasing the driving force of its oxidation. Surprisingly, the relative amplitudes of the phases reflecting PhQ(A)(*-) and PhQ(B)(*-) oxidation were also affected by these mutations: the apparent PhQ(A)(*-)/PhQ(B)(*-) ratio is shifted from 0.65:0.35 in wild-type reaction centers to 0.5:0.5 in PsaA-L722Y/T and to 0.8:0.2 in PsaB-L706Y. The most consistent account for all these observations involves considering reversibility of oxidation of PhQ(A)(*-) and PhQ(B)(*-) by F(X), and asymmetry in the driving forces for these electron transfer reactions, which in turn leads to F(x)-mediated interquinone electron transfer.

Elevated proton leak of the intermediate OH in cytochrome c oxidase.

The kinetics of the formation and relaxation of transmembrane electric potential (Deltapsi) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa(3)-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Deltapsi kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O(H)) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Deltapsi (tau(app) < or = 0.5-0.8 s). By contrast, when the enzyme in the O(H) state is rapidly re-reduced by sequential electron delivery, Deltapsi dissipates much slower (tau(app) > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Deltapsi dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O(H) state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.

Additive effect of mutations affecting the rate of phylloquinone reoxidation and directionality of electron transfer within photosystem I.

Optical pump-probe spectroscopy in the nanosecond-microsecond timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA-Y696F) near the primary acceptor chlorophyll, ec3A, with those near PhQA (PsaA-S692A, PsaA-W697F). The PsaA-S692A and PsaA-W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild-type (WT). The reverse similar 20 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a reverse similar 20 ns component (PhQB(-) reoxidation) with increased amplitude compared with the WT and the individual PhQA mutants. The component assigned to PhQA(-) reoxidation was slowed, like the individual PhQA mutants, and of lower amplitude, as observed in the single ec3A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the reverse similar 20 and reverse similar 200 ns phases to reoxidation of PhQB or PhQA, respectively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (approximately 180 ns) component, as previously reported for mutants of the PhQ(A) binding pocket (Biochim. Biophys. Acta [2006] 1757, 1529-1538), which we have tentatively attributed to forward electron transfer between the iron-sulfur clusters FX and FA/B.

Nanosecond electron tunneling between the hemes in cytochrome bo3.

Biological electron transfer (eT) between redox-active cofactors is thought to occur by quantum-mechanical tunneling. However, in many cases the observed rate is limited by other reactions coupled to eT, such as proton transfer, conformational changes, or catalytic chemistry at an active site. A prominent example of this phenomenon is the eT between the heme groups of mitochondrial cytochrome c oxidase, which has been reported to take place in several different time domains. The question of whether pure eT tunneling in the nanosecond regime between the heme groups can be observed has been the subject of some experimental controversy. Here, we report direct observations of eT between the heme groups of the quinol oxidase cytochrome bo(3) from Escherichia coli, where the reaction is initiated by photolysis of carbon monoxide from heme o(3). eT from CO-dissociated ferrous heme o(3) to the low-spin ferric heme b takes place at a rate of (1.2 ns)(-1) at 20 degrees C as determined by optical spectroscopy. These results establish heme-heme electron tunneling in the bo(3) enzyme, a bacterial relative to the mitochondrial cytochrome c oxidase. The properties of eT between the closely lying heme groups in the heme-copper oxidases are discussed in terms of the reorganization energy for the process, and two methods for assessing the rate of electron tunneling are presented.

In vivo multiphoton nanosurgery on cortical neurons.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus.

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy.

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

Plasticity of proton pathway structure and water coordination in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

Ligand dynamics in an electron transfer protein. Picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c.

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin.

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.

Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL.

FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme pocket as a second step in the signal pathway on the 50 ps time scale.

Activationless electron transfer through the hydrophobic core of cytochrome c oxidase.

Electron transfer (ET) within proteins occurs by means of chains of redox intermediates that favor directional and efficient electron delivery to an acceptor. Individual ET steps are energetically characterized by the electronic coupling V, driving force DeltaG, and reorganization energy lambda. lambda reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; lambda approximately 700-1,100 meV (1 eV = 1.602 x 10(-19) J) has been considered as a typical value for intraprotein ET. In nonphotosynthetic systems, functional ET is difficult to assess directly. However, using femtosecond flash photolysis of the CO-poised membrane protein cytochrome c oxidase, the intrinsic rate constant of the low-DeltaG electron injection from heme a into the heme a(3)-Cu(B) active site was recently established at (1.4 ns)(-1). Here, we determine the temperature dependence of both the rate constant and DeltaG of this reaction and establish that this reaction is activationless. Using a quantum mechanical form of nonadiabatic ET theory and common assumptions for the coupled vibrational modes, we deduce that lambda is <200 meV. It is demonstrated that the previously accepted value of 760 meV actually originates from the temperature dependence of Cu(B)-CO bond breaking. We discuss that low-DeltaG, low-lambda reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins.

Electron transfer between hemes in mammalian cytochrome c oxidase.

Fast intraprotein electron transfer reactions associated with enzymatic catalysis are often difficult to synchronize and therefore to monitor directly in non-light-driven systems. However, in the mitochondrial respiratory enzyme cytochrome oxidase aa(3), the kinetics of the final electron transfer step into the active site can be determined: reverse electron flow between the close-lying and chemically identical hemes a(3) and a can be initiated by flash photolysis of CO from reduced heme a(3) under conditions where heme a is initially oxidized. To follow this reaction, we used transient absorption spectroscopy, with femtosecond time resolution and a time window extending to 4 ns. Comparison of the picosecond heme a(3)-CO photodissociation spectra under different redox states of heme a shows significant spectral interaction between both hemes, a phenomenon complicating the interpretation of spectral studies with low time resolution. Most importantly, we show that the intrinsic electron equilibration, corresponding to a DeltaG(0) of 45-55 meV, occurs in 1.2 +/- 0.1 ns. This is 3 orders of magnitude faster than the previously established equilibration phase of approximately 3 mus, which we suggest to reflect a change in redox equilibrium closely following CO migration out of the active site. Our results allow testing a number of conflicting predictions regarding this reaction between both experimental and theoretical studies. We discuss the potential physiological relevance of fast equilibration associated with this low-driving-force redox reaction.

The catalytic cycle of cytochrome c oxidase is not the sum of its two halves.

Membrane-bound cytochrome c oxidase catalyzes cell respiration in aerobic organisms and is a primary energy transducer in biology. The two halves of the catalytic cycle may be studied separately: in an oxidative phase, the enzyme is oxidized by O(2), and in a reductive phase, the oxidized enzyme is reduced before binding the next O(2) molecule. Here we show by time-resolved membrane potential and pH measurements with cytochrome oxidase liposomes that, with both phases in succession, two protons are translocated during each phase, one during each individual electron transfer step. However, when the reductive phase is not immediately preceded by oxidation, it follows a different reaction pathway no longer coupled to proton pumping. Metastable states with altered redox properties of the metal centers are accessed during turnover and relax when external electron donors are exhausted but recover after enzyme reduction and reoxidation by O(2). The efficiency of ATP synthesis might be regulated by switching between the two catalytic pathways.

Proton and electron pathways in the bacterial nitric oxide reductase.

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.