Bulk Water Phase Research Articles

Исследование распределения ингибиторов коррозии между водной и нефтяной фазами

Partitioning behaviours of commercial inhibitors like ethoxylated amine and phosphate ester were studied by carrying out partitioning tests and determining the concentration of the inhibitors by conventional colorimetry method and Inductively Coupled Plasma spectroscopy. The tests were performed using various water cuts, hydrocarbon phases, and concentrations in order to find out the effects of these factors. Ethoxylated amine distributed itself mostly into aqueous phase regardless of the conditions. Comparison of partitioning behaviour of phosphate ester in low and high water cut mixtures resulted in some loss of the inhibitor from bulk water phase as water cut decreased. Phosphate ester inhibitor showed significant synergistic effect with other nitrogenous compounds during the partitioning experiments.

Changes in biofilm composition and microbial water quality in drinking water distribution systems by temperature increase induced through thermal energy recovery

Drinking water distribution systems (DWDSs) have been thoroughly studied, but the concept of thermal energy recovery from DWDSs is very new and has been conceptualized in the past few years. Cold recovery results in a temperature increase of the drinking water. Its effects on drinking water quality and biofilm development are unclear. Hence, we studied both bulk water and biofilm phases for 232 days in two parallel pilot scale distribution systems with two temperature settings after cold recovery, 25 °C and 30 °C, and compared these with a reference pilot system without cold recovery. In all three pilot distributions systems (DSs) our results showed an initial increase in biomass (ATP) in the biofilm phase, along with occurrence of primary colonizers (Betaproteobacteriales) and subsequently a decrease in biomass and an increasing relative abundance of other microbial groups (amoeba resisting groups; Xanthobacteraceae, Legionellales), including those responsible for EPS formation in biofilms (Sphingomonadaceae). The timeline for biofilm microbial development was different for the three pilot DSs: the higher the temperature, the faster the development took place. With respect to the water phase within the three pilot DSs, major microbial contributions came from the feed water (17–100%) and unkown sources (2–80%). Random contributions of biofilm (0–70%) were seen between day 7–77. During this time period six-fold higher ATP concentration (7–11 ng/l) and two-fold higher numbers of high nucleic acid cells (5.20–5.80 × 104 cells/ml) were also observed in the effluent water from all three pilot DSs, compared to the feed water. At the end of the experimental period the microbial composition of effluent water from three pilot DSs revealed no differences, except the presence of a biofilm related microbial group (Sphingomonadaceae), within all three DSs compared to the feed water. In the biofilm phase higher temperatures initiated the growth of primary colonizing bacteria but this did not lead to differences in microbial diversity and composition at the end of the experimental period. Hence, we propose that the microbiological water quality of DWDSs with cold recovery should be monitored more frequently during the first 2–3 months of operation.

Effect of trehalose on protein cryoprotection: Insights into the mechanism of slowing down of hydration water.

We study, with molecular dynamics simulations, a lysozyme protein immersed in a water-trehalose solution upon cooling. The aim is to understand the cryoprotectant role played by this disaccharide through the modifications that it induces on the slow dynamics of protein hydration water with its presence. The α-relaxation shows a fragile to strong crossover about 20° higher than that in the bulk water phase and 15° higher than that in lysozyme hydration water without trehalose. The protein hydration water without trehalose was found to show a second slower relaxation exhibiting a strong to strong crossover coupled with the protein dynamical transition. This slower relaxation time importantly appears enormously slowed down in our cryoprotectant solution. On the other hand, this long-relaxation in the presence of trehalose is also connected with a stronger damping of the protein structural fluctuations than that found when the protein is in contact with the pure hydration water. Therefore, this appears to be the mechanism through which trehalose manifests its cryoprotecting function.

The increased viscosity effect for fracturing fluid imbibition in shale

Understanding water imbibition behaviors in shale formations plays an essential role in shale gas development. The effective viscosity of water in shale nanopores is usually different from that of the bulk phase because of confined conditions. In this work, a model considering interactions between water and a solid surface is proposed to predict the effective viscosity of water at the nanoscale, which is inserted into the classic Lucas-Washburn (L-W) model to describe the water imbibition behavior in shale formations. This model is derived based on a molecular kinetic theory and incorporates the disjoining pressure. Published experimental data is used to validate the model. It is demonstrated that the effective viscosity of water in hydrophilic nanopores is much higher than that of the bulk phase because of strong interactions between water and a solid surface, and the deviation significantly increases when the separation is below 10 nm. For a hydrophilic capillary tube with a diameter of 10 nm, the water effective viscosity is approximately 2.5 times higher than that of the bulk phase. Moreover, this deviation is larger for a capillary tube compared with a capillary channel, because of the curvature effect. Besides, the effective viscosity for water under a hydrophobic condition is smaller than that of the bulk phase water, because the structural repulsive force dominates under a hydrophobic condition. This work establishes a theoretical foundation to calculate the effective viscosity for water flow at the nanoscale. Furthermore, it helps to understand fracturing fluid imbibition behavior in shale gas reservoirs, which will benefit the simulation of fluid flow at the reservoir scale.

Tubules, beads, discs and junctions – Morphologies and dynamics of dispersed multilamellar lipid phases in excess water

In this paper, experimental results on the swelling, dispersion and disintegration of the lamellae composed of the lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in contact with excess water are described. Multilamellar structures nucleate and grow at the interface between the pure solvent and the lipid. The system evolves slowly via flow and coalescence of lamellae through a variety of non-equilibrium morphologies demonstrating that their dynamics is complex and non-universal, with no unique kinetic pathway. The tubular structures disintegrate slowly into the bulk water phase, inside the sealed sample chamber.

Controlling the Emulsion Type Using Adjustable Polyelectrolyte-Surfactant Complexes.

The combination of polyelectrolytes and ionic surfactants in precise proportions presents the possibility of producing a new class of emulsifiers with tunable emulsification properties. We use chitosan along with dioctyl sulfosuccinate sodium, also known as aerosol-OT (AOT), to demonstrate that emulsion types can be varied, and phase inversion emulsification (PIE) can be induced via changes in the water-phase pH and the molar ratio of the surfactant to the repeat unit of the polyelectrolyte. Confocal microscopy of the emulsions shows that the morphology can be changed from O/W to O/W/O to W/O by varying the surfactant to polyelectrolyte molar ratio at a fixed aqueous-phase pH while maintaining droplet sizes in the range of micrometers to tens of micrometers. Measurements of the oil (toluene)-water partition coefficient suggest that controlling the emulsion type relies on the ability of the surfactants to partition from the bulk oil to the bulk water phase and to induce polyelectrolyte-surfactant aggregation. We confirm this hypothesis using different combinations of polyelectrolytes and surfactants. Changes in the water-phase pH in situ induce phase inversion only in a particular direction, which suggests that the complexes at the interface are in a kinetically trapped state. Changes in the molar ratio in situ by addition of an oppositely charged surfactant also can induce phase inversion.

Origin of chronoamperometric responses associated with impacts of single electrolyte droplets at a polarized liquid/liquid interface

Chronoamperometric measurements at a constant potential are used to study the single impacts of the water-in-DCE emulsion droplets containing an electrolyte (tetrapropylammonium chloride, TPrACl) at a polarized water(W)/1,2-dichloroethane (DCE) interface. The impacts give rise to the spike-like current transients, which are characterized by the initial current step (time constant less than 2 ms) followed by a slower exponential current decay with a time constant in tens of milliseconds. It is shown that the theoretical treatment of the current spikes can be based on a modification of the bulk electrolysis model including (1) the droplet collision at the W/DCE interface, (2) a partial coalescence (fusion) of the droplet with the impacted interface, (3) a reorganization of the electric double layer leading to the propagation of the interfacial potential difference from the impacted interface to the surface of the droplet, and (4) stripping (bulk electrolysis) of TPrA+ from the droplet to the DCE phase accompanied by the chloride depletion into the bulk water phase. On assuming that the stripping of TPrA+ is the rate-determining step of the impact event, the theoretical relationships for the current transient are derived, which are confirmed by the experimental data. An analysis of the current transients yields the charge corresponding to the ionic content of the droplets, which is used to evaluate the droplet diameter. Droplet size distribution with the droplet diameter of around 8 μm is verified by an analysis of the microscopic images of the W-in-DCE emulsion, and in part also by the dynamic laser scattering measurements. It is shown that the frequency of the droplet collisions with the W/DCE interface (1–10 Hz) is controlled by migration of the negatively charged droplets towards the interface.

Osmotic pressure effects identify dehydration upon cytochrome c-cytochrome c oxidase complex formation contributing to a specific electron pathway formation.

In the electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO), we determined the number and sites of the hydration water released from the protein surface upon the formation of the ET complex by evaluating the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO. We identified that ∼20 water molecules were dehydrated in complex formation under turnover conditions, and systematic Cyt c mutations in the interaction site for CcO revealed that nearly half of the released hydration water during the complexation were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a dehydration dominantly compensates for the entropy decrease due to the association of Cyt c with CcO, resulting in the entropy-driven ET reaction. The energetic analysis of the interprotein interactions in the ET complex predicted by the docking simulation suggested the formation of hydrophobic interaction sites surrounding the exposed heme periphery of Cyt c in the Cyt c-CcO interface (a 'molecular breakwater'). Such sites would contribute to the formation of the hydrophobic ET pathway from Cyt c to CcO by blocking water access from the bulk water phase.

Molecular Mobility Suppression of Ibuprofen-Rich Amorphous Nanodroplets by HPMC Revealed by NMR Relaxometry and Its Significance with Respect to Crystallization Inhibition

In the present study, the molecular state of drug-rich amorphous nanodroplets was evaluated using NMR techniques to reveal the mechanism underlying the crystallization inhibition of drug-rich amorphous nanodroplets by a polymer. Ibuprofen (IBP) with a low glass transition temperature was used for direct characterization of drug-rich amorphous nanodroplets. Highly supersaturated IBP formed IBP-rich amorphous nanodroplets through phase separation from aqueous solution. Increasing the concentration of hypromellose (HPMC) in the aqueous solution contributed to the inhibition of IBP crystallization and maintenance of supersaturation at IBP amorphous solubility. Solution 1H NMR measurements of IBP supersaturated solution containing IBP-rich amorphous nanodroplets clearly showed two kinds of 1H peaks derived from the dissolved IBP in bulk water phase and phase-separated IBP in IBP-rich amorphous nanodroplets. NMR spectral analysis indicated that HPMC did not affect the chemical environment and mobility of the dissolved IBP. However, 1H spin-spin relaxation time measurements clarified that the dissolved IBP in the bulk water phase was exchanged with the IBP-rich amorphous nanodroplets with an exchange lifetime of more than 10 ms. Moreover, the 1H peaks of HPMC partially disappeared due to the formation of IBP-rich amorphous nanodroplets, suggesting that a part of HPMC distributed into the IBP-rich amorphous nanodroplets from the bulk water phase. The incorporation of HPMC significantly changed the chemical environment of the phase-separated IBP in the IBP-rich amorphous nanodroplets and strongly suppressed molecular mobility. The resulting molecular mobility suppression effectively inhibited IBP crystallization from the IBP-rich amorphous nanodroplets. Thus, direct investigation of drug-rich amorphous nanodroplets using NMR can be a promising approach for selecting appropriate pharmaceutical excipients to suppress drug crystallization in supersaturated drug solutions.

Bulk phase water diffusion is significantly inhibited by inhomogeneity of single non-crystal particle at low relative humidity

It has been suggested by recent studies that atmospheric particles adopt non-crystalline states which significantly impact aerosol-cloud interactions and atmospheric chemistry. In this study, the effect of non-crystalline states on water diffusion is detailed investigated from single multi-component particles levitated in aerosol optical tweezers. We infer the time-dependent particle size from Raman spectra using Mie fitting, thus derive the water diffusion coefficient (Dwater) from particle radius changes during evaporation or condensation processes. In both glassy states (in saccharide particles) and gel states (in MgSO4 particles), the bulk phase water diffusion is shown to be severely restricted, thus limiting the gas-particle water partitioning on the particle surface. The Dwater of glassy particles generally gradually decreases as the RH decreases, while the relative humidity (RH) - Dwater relationship of particle in gel state is complicated and brings huge deviation of Dwater determination. We therefore present the time dependent water content at different location (radial coordinate) of the particle. The time scale required for particle to get equilibrium to environmental RH is vastly extended by the kinetic inhibition of bulk phase water transfer. This can give direct and quantitative indication of water diffusion within single non-crystalline particle and its effect on gas-particle partitioning and equilibrium.

MemBlob database and server for identifying transmembrane regions using cryo-EM maps.

SummaryThe identification of transmembrane helices in transmembrane proteins is crucial, not only to understand their mechanism of action but also to develop new therapies. While experimental data on the boundaries of membrane-embedded regions are sparse, this information is present in cryo-electron microscopy (cryo-EM) density maps and it has not been utilized yet for determining membrane regions. We developed a computational pipeline, where the inputs of a cryo-EM map, the corresponding atomistic structure, and the potential bilayer orientation determined by TMDET algorithm of a given protein result in an output defining the residues assigned to the bulk water phase, lipid interface and the lipid hydrophobic core. Based on this method, we built a database involving published cryo-EM protein structures and a server to be able to compute this data for newly obtained structures.Availability and implementation http://memblob.hegelab.org.Supplementary information Supplementary data are available at Bioinformatics online.

Molecular dynamics simulation of interface-mediated GO-GO interaction at the air-water interface

Developing self-assembly of two-dimensional GO nanosheets at the air-liquid interface requires the knowledge of how interfaces affect the effective GO-GO interaction. In this work, we use molecular dynamics simulation to compute the potentials of mean force between two GO sheets in the vicinity of the air-water interface. Our simulation result shows that the thermodynamic interaction between GOs is enhanced in the interface, suggesting that GO aggregation at the air-liquid interface is more favorable than that in the bulk. The air-water interface environment can lead to reduced repulsive solvent-induced solvation force, and, more specifically, decreases the interlayer solvent effect, as compared with the bulk water phase. It is further found that GO sheets with different oxidation degrees demonstrate diverse colloidal kinetics stability in the interface. In particular, GO sheets with high oxidation degree exhibit similar stability both at the interface and in the bulk phase, which is attributed to the enhanced hydrogen-bonding interaction between interlayer water molecules and oxygen functional groups. The interface-mediated GO-GO interaction, revealed in this work, provides new insight into the effect of the air-water interface on the self-assembly processes of two-dimensional GO nanosheets.

Study on ethane hydrate formation/dissociation in a sub-millimeter sized capillary

Study on the gas hydrates in porous medium has great significance to the formation, accumulation and exploitation of natural gas hydrate (NGH). Consequently, we investigated the formation/dissociation of ethane hydrate in a sub-millimeter sized capillary in this work, and observed the appearance and kinetic rate during the hydrate formation/dissociation process. The dissociation equilibria of ethane hydrate in capillary was measured first, then the influences of hydrate memory effect (dissociation temperature and time) and degree of subcooling on ethane hydrate formation were obtained, respectively. The results demonstrate that hydrate forms more difficultly in capillary than in conventional macro-reactor because of the fluid resistance effect and poor mass transfer of gas-liquid. The appearance and formation kinetics of ethane hydrate are greatly influenced by the memory effect and subcooling degree. The morphology of ethane hydrate formation on the bubble surface in water bulk phase shows that “bridge effect” occurs during the hydrate formation and growth in capillary.

Molecular dynamics characterization of the water-methane, ethane, and propane gas mixture interfaces

The co-existing natural gas and water bulk phases arise in a wide range of technological and environmental processes. The liquid-gas mixture is separated by an interface which plays a crucial role in mass transport across the phases. In this work, we use the molecular dynamics (MD) technique to investigate the molecular organization, solubility, density, and compositions of natural gas-water interfaces. We apply the NPNAT ensemble which is an appropriate statistical methodology to dodge the defects of the conventional ensembles in surface physics, and ultimately, to characterize the interfacial thermodynamics and mechanics. High interfacial density, excess, and radial pair distribution function of the gas components in order of propane, ethane, and methane, respectively, suggest the interfacial adsorption as per favorable interactions with a dense hydrogen bonding network near the surface in the liquid water phase. It is also found that the gas components solubility is negligible. Nevertheless, methane molecules present in natural gas can further dissolve in water, comparing to pure methane. We lastly conclude a heterogeneous formation of structure II hydrate from the adsorption and composition results. Moreover, we systematically increase the pressure from 1 MPa to 50 MPa and the temperature from 273.15 K to 303.15 K to calculate the interfacial tension using the mechanical approach. We observe a decrease in the interfacial tension along with an increase in both pressure and temperature. Given the remarkable hydrocarbon adsorption acting as a surfactant, this interfacial tension attenuation is more highlighted in a natural gas-water system compared to the pure liquid-vapor water or water-pure methane systems at the same temperature and pressure. We employ MD combined with fundamental thermodynamics to predict the interfacial tension via its independent relations with pressure and temperature which agrees with the classical scaling laws, namely, the Eötvös rule. The corresponding molecular mechanisms captured by the microscopic and macroscopic properties at the interfacial regions prospectively demonstrate a sensitivity to both temperature and pressure, which contributes to the developing understanding and applications of the imperative water-natural gas interface.

Molecular dynamics simulations of surfactant adsorption at oil/water interface under shear flow

Surfactants are extensively used in many chemical products to improve their stability, appearance, texture, and rheology. Precise control of the emulsion droplet size distribution, which depends on the characteristics of the surfactant used, is important for target-oriented product design. A complete understanding of the structures and dynamics of emulsion droplets at the reactor level requires coupling of two mesoscale physical constraints, that at the interfacial level, i.e., smaller than a single droplet (Mesoscale-1), and that at the device level, i.e., larger than a single droplet (Mesoscale-2). In this work, the structures and adsorption kinetics of Mesoscale-1 surfactant molecules were studied via coarse-grained molecular dynamics. A non-equilibrium model that could introduce stable shear flow into the simulation box was used to investigate the interfacial structures at the droplet interface under different shear rates. The configurations of the surfactant molecules and adsorption amounts were compared with those obtained without flow. The adsorption kinetics for different shear rates were compared to determine the effects of hydrodynamic interactions. The dominant mechanisms governing the dynamic structures can thus be summarized as maximization of the adsorption density at the interface and minimization of flow resistance in the bulk phase (water and/or oil molecules). A scheme for coupling between Mesoscale-1 and Mesoscale-2 is proposed. This method is promising for the incorporation of interfacial structure effects into the hydrodynamics at the reactor device level for the manipulation of chemical products.

Effect of Hydrophobic Silica Nanoparticles on the Kinetics of Methane Hydrate Formation in Water-in-Oil Emulsions

This study addresses the effect of hydrophobic silica nanoparticles (SiNPs) on methane hydrate formation in water-in-decane emulsions. Even though the new concept of hydrophobic particle inhibitors has been proposed in the flat interface of oil and bulk water phases, the mechanism of the particle inhibition has not been investigated for a hydrate-forming emulsion system. Numerous experiments with various amounts of SiNPs applied to water-in-decane emulsions and different temperature and pressure profiles have verified how the nanoparticles interact at the interface for hydrate inhibition or promotion. At lower concentrations of SiNPs, they act as a hydrate inhibitor. However, as the amount of SiNPs increases beyond a certain concentration (0.5 wt %), the formation rate anomalously increases because the oil film of the emulsions containing methane becomes thicker with the increasing amount of SiNPs, and water molecules form capillary bridges along the nanoparticle alignment. Our experimental results and su...

Experimental investigations of methane hydrate in sediment suspensions

Methane hydrate structural stability and formation, were studied in water suspensions with synthetic clay (44.17nm),SiO2(60.62nm) and zeolite (35.08nm) at different weight percentages (1wt%,10wt%,20wt%) in isochoric method using stirred autoclave. The for- mation kinetics in these sediments greatly differs from those of the bulk hydrate owing to surface and capillary effects in the pores. It has been confirmed that hydrate formation in confined pores is severely restricted due to increased inner capillary pressure. Further- more in principle , the clay particles suspended in water play a negative role in structuring the crystalline hydrate .More specifically, even though the addition of clay enhances the hydrate yield insignificantly, from our results, the addition of sediments did not affect the phase equilibrium much because water exists in excess around clay particles and therefore the hydrate formed in the bulk water phase is much more dominant than that in the pores.

Rates of cavity filling by liquids

Understanding the fundamental wetting behavior of liquids on surfaces with pores or cavities provides insights into the wetting phenomena associated with rough or patterned surfaces, such as skin and fabrics, as well as the development of everyday products such as ointments and paints, and industrial applications such as enhanced oil recovery and pitting during chemical mechanical polishing. We have studied, both experimentally and theoretically, the dynamics of the transitions from the unfilled/partially filled (Cassie-Baxter) wetting state to the fully filled (Wenzel) wetting state on intrinsically hydrophilic surfaces (intrinsic water contact angle <90°, where the Wenzel state is always the thermodynamically favorable state, while a temporary metastable Cassie-Baxter state can also exist) to determine the variables that control the rates of such transitions. We prepared silicon wafers with cylindrical cavities of different geometries and immersed them in bulk water. With bright-field and confocal fluorescence microscopy, we observed the details of, and the rates associated with, water penetration into the cavities from the bulk. We find that unconnected, reentrant cavities (i.e., cavities that open up below the surface) have the slowest cavity-filling rates, while connected or non-reentrant cavities undergo very rapid transitions. Using these unconnected, reentrant cavities, we identified the variables that affect cavity-filling rates: (i) the intrinsic contact angle, (ii) the concentration of dissolved air in the bulk water phase (i.e., aeration), (iii) the liquid volatility that determines the rate of capillary condensation inside the cavities, and (iv) the presence of surfactants.

Combined effects of the drug distribution and mucus diffusion properties of self-microemulsifying drug delivery systems on the oral absorption of fenofibrate

We present the absorption improvement mechanism of fenofibrate (FFB), a Biopharmaceutics Classification System (BCS) class II drug, from self-microemulsifying drug delivery systems (SMEDDS), centered on improving the diffusion of FFB through the unstirred water layer (UWL). Four SMEDDS formulations containing Labrafac™ lipophile WL 1349 (WL1349) or Labrafil® M 1944CS (M1944) oils and NIKKOL HCO-40 (HCO40) or NIKKOL HCO-60 (HCO60) surfactants were prepared. Every SMEDDS formulation formed microemulsion droplets of approximately 30 nm. In vitro tests showed that the microemulsion droplets containing M1944 had relatively small FFB solubilization capacities, causing larger amounts of FFB to be dissolved in the bulk water phase, compared to the droplets containing WL1349. The diffusivity of the microemulsion droplets through the mucin solution layer was enhanced when using HCO40 compared to HCO60. The oral absorption in rats was the highest when using the SMEDDS formulation containing M1944 and HCO40. High FFB distribution in the bulk water phase and fast diffusion of microemulsion droplets through the mucus layer contributed to the efficient delivery of FFB molecules through the UWL to the epithelial cells, leading to enhanced FFB absorption.

Electron Transfer Pathway Analysis from Cytochrome C to Cytochrome C Oxidase Under Turnover Conditions

The electron transfer (ET) reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. A series of ET reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water. Associated with the reduction of molecular oxygen, CcO functions as a proton pump across the membrane, and the proton gradient is the primary driving force for the generation of ATP. In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at CcO are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c forms an ET complex with CcO to promote the ET reaction. While the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO to form the ET pathway from Cyt c to CcO. To identify the specific interactions for the complex formation between Cyt c and CcO and estimate the ET pathway, we have determined the interaction site of Cyt c for CcO using NMR spectroscopy1. However, the NMR measurements can be applied only for ET complexes such as oxidized Cyt c – fully oxidized CcO or reduced Cyt c – fully reduced CcO complexes, where the ET reaction does not occur. In addition, our previous NMR analysis1 identified the CcO interaction site on Cyt c, while no information about the Cyt c interaction site on CcO has been obtained. A detailed analysis of the interactions essential for the formation of the ET pathway in the ET complexes under turnover conditions, where the ET reaction from Cyt c to CcO is induced, has not yet been conducted. Based on the mutational effects on the steady state kinetics of the ET reaction and our NMR analysis of the interaction site, we examined the structure of the ET complex between Cyt c and CcO under turnover conditions using protein docking simulation2, and energetically characterized the interactions essential for complex formation and estimated the ET pathway from Cyt c to CcO. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the ET reaction to CcO. The interaction analysis using the selected Cyt c – CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions which were almost cancelled out by large favorable (Columbic) electrostatic interactions, but resulting in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, while the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. To determine the ET pathway from Cyt c to CcO, the pathway analysis3 was applied to the predicted ET complex between Cyt c and CcO under turnover conditions. The estimated ET pathway was found to be constructed by many hydrophobic amino acid residues, including Trp104 of the subunit II of CcO, which is supposed to be the electron entry site for CcO from Cyt c. The contribution of many hydrophobic amino acid residues to formation of the ET pathway also supports the crucial role of the dehydration from hydrophobic amino acid residues associated with the complex formation between Cyt c and CcO, and suggests formation of a structure blocking water access from the bulk water phase (a “molecular breakwater”) surrounding the ET pathway across the Cyt c - CcO interface to avoid formation of hydrogen-bond mediated non-specific ET pathways. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., Proc. Natl. Acad. Sci. USA 2011, 108, 12271-12276.Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., J. Biol. Chem. 2016, 291, 15320-15331.Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Ann. Rev. Biophys. Biomol. Struct. 1992, 21, 349-377.