Reorganization energies of the electron transfer reactions involving quinones in the reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In framework of the continuum electrostatics theory, the reorganization energies of the electron transfers Q_A^?–Q_B (fast phase), Bph^?–Q_A, P⁺–Q_A^?, and P⁺–Q_B^? in the photosynthetic bacterial reaction center have been calculated. The calculations were based on the static dielectric permittivity spatial distribution derived from the data on the electrogenesis, with the corresponding characteristic times relatively close to the reaction times of Q_A^?–Q_B (fast phase) and Bph^?–Q_A but much shorter than those times of the latter two recombination reactions. The calculated reorganization energies were reasonably close to the experimental estimates for Q_A^?–Q_B (fast phase) and Bph^?–Q_A but substantially lower than those of P⁺–Q_A^? and P⁺–Q_B^?. A higher effective dielectric permittivity contributes to this effect, but the dominant contribution is most probably made by a non-dielectric relaxation, especially for the P⁺–Q_B^? recombination influenced by the proton transfer. This situation calls for reconsidering of the current electron transfer rate estimates.     

Delayed fluorescence study on P*QA→ P+QA − charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P⁺Q_A– relative to that of P–Q_A was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–Q_A and P⁺Q_A– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P⁺Q_A– state runs parallel to the (integrated) net proton uptake due to the PQ_A/P⁺Q_A– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pK_a values as well as the light-induced pK_a changes of the protonatable groups of the protein. The observed dependence of the energetics of P⁺Q_A– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Effects of the high-molecular-weight cryoprotectant dextran on fluid secretion by Calliphora salivary glands

The rate of fluid secretion by isolated salivary glands of Calliphora was inhibited as a linear function of dextran and poly vinyl pyrrolidone concentrations in the range 15–35% w/w. This inhibition was not overcome by supramaximal concentrations of 5-hydroxytryptamine, nor was it caused by a decreased availability of K⁺ from the medium. Although the polymers caused large decreases of freezing point (and vapor pressure) of the incubation medium, the glands did not respond to this by secreting a more K ⁺-rich saliva. When dextran and polyvinyl pyrrolidone were added as powders to salt solutions, the total freezing-point depression of the mixture was equal to the sum of that exerted by the pure salt solution and that expected for the polymer concentration. The activities of K⁺ and Cl^?, as measured by ion-selective electrodes, were not increased in solutions by the addition of dextran. Dextran was demonstrated by electron microscopy to penetrate into the basal clefts and intercellular spaces of the isolated glands. These results demonstrate that addition of dextran (and probably of polyvinyl pyrrolidone) does not decrease the solvent activity of water in physiological salt solutions. The inhibition of fluid secretion by isolated salivary glands of Calliphora seems therefore due only to the altered physical characteristics of the medium.     

Biochemistry of microbial polyvinyl alcohol degradation

Effect of minor chemical structures such as 1,2-diol content, ethylene content, tacticity, a degree of polymerization, and a degree of saponification of the main chain on biodegradability of polyvinyl alcohol (PVA) is summarized. Most PVA-degraders are Gram-negative bacteria belonging to the Pseudomonads and Sphingomonads, but Gram-positive bacteria also have PVA-degrading abilities. Several examples show symbiotic degradation of PVA by different mechanisms. Penicillium sp. is the only reported eukaryotic degrader. A vinyl alcohol oligomer-utilizing fungus, Geotrichum fermentans WF9101, has also been reported. Lignolytic fungi have displayed non-specific degradation of PVA. Extensive published studies have established a two-step process for the biodegradation of PVA. Some bacteria excrete extracellular PVA oxidase to yield oxidized PVA, which is partly under spontaneous depolymerization and is further metabolized by the second step enzyme (hydrolase). On the other hand, PVA (whole and depolymerized to some extent) must be taken up into the periplasmic space of some Gram-negative bacteria, where PVA is oxidized by PVA dehydrogenase, coupled to a respiratory chain. The complete pva operon was identified in Sphingopyxis sp. 113P3. Anaerobic biodegradability of PVA has also been suggested.     

Free energy change accompanying electron transfer from P870 to quinone in Rhodopseudomonas sphaeroides chromatophores

Delayed fluorescence of chromatophores of Rhodopseudomonas sphaeroides was measured to estimate the standard free energy change accompanying the electron transfer from the bacteriochlorophyll dimer (P) to the primary acceptor quinone (Q_A). The chromatophores emitted delayed fluorescence with a lifetime of about 60 ms in the presence of o-phenanthroline. By comparing the intensity of the delayed fluorescence with that of the prompt fluorescence, the standard free energy of the P⁺Q_A^? radical pair was evaluated. It was about 0.87 eV below the level of excited singlet state, P1Q_A, or 0.51 eV above the ground state, PQ_A, independent of pH.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

Effect of hydrogen bonds on the energetics of macromolecules in the course of electron transfer

For a model system consisting of a bacteriochlorophyll dimer (P) and a primary quinone with the nearest environment (Q_A), which are the electron donor and acceptor in the recombination reaction in the Rhodobacter spheroides reaction center, the energies of states P⁺Q _A ^? and PQ_A have been calculated at several stable conformations of Q_A that differ in the positions of the proton involved in the hydrogen bond. It is shown that the position of the proton has a considerable influence on the energy of vertical transition P⁺Q _A ^? → PQ_A.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris

The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K _cat /K _m of recombinant OPH was 598 mM^?1 s^?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.     

Degradation of polyvinyl alcohol by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. SA3 and its symbiote

A total of 800 samples was taken from Taegu province, Korea, where many textile factories provide a source of polyvinyl alcohol (PVA) waste. These samples were screened for PVA-degrading bacteria. A new strain, SA3, was discovered which formed yellow colonies and used PVA as the sole carbon and energy source. Strain SA3 was identified as a Sphingomonas sp., based on the partial nucleotide sequence analysis of 16S ribosomal RNA, the presence of 2-hydroxymyristic acid (14:O 2-OH) and sphingolipids with d-17:0, d-18:0, d-19:1, and d-20:1 as the main dihydrosphingosines. This genus has not previously been reported as a PVA-degrading bacterium. Sphingomonas sp. SA3 needs a symbiote strain, SA2, for PVA degradation as a growth factor producer. In mixed cultures of these strains, the optimum temperature for PVA biodegradation ranged from 30 °C to 35 °C. The optimum pH was 8.0 and the most effective nitrogen source was NH₄ ⁺. Electronic Publication     

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS1

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O₂ evolution and inhibited the water‐splitting system in PSII. S‐states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSII_X) and PSII_β reaction centers, which increased the fluxes of dissipated energy. JIP test and Q_A^? reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from Q_A^? to Q_B/Q_B^? and accumulation of P₆₈₀⁺. More Q_A^? had to be oxidized through S₂(Q_AQ_B)^? charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.     

Phenanthrene biodegradation by immobilized microbial consortium in polyvinyl alcohol cryogel beads

Removal of polycyclic aromatic hydrocarbons (PAHs), a group of widespread toxic compounds, has been one of the environmental issues in wastewater treatment systems for many years. In this study, biodegradation of phenanthrene (PHE), as a model contaminant, by a microbial consortium entrapped in polyvinyl alcohol (PVA) cryogel prepared by freeze-thaw method was investigated. The effect of inoculum size (300–900 mg of cell dry weight per liter) and initial PHE concentration (100–2000 ppm) as well as bead cell density (5 and 10 mg ml⁻¹) on PHE biodegradation by freely suspended cell (FC) and immobilized cell (IC) systems in aqueous phase was examined. Results showed that although both IC and FC systems were capable of complete removal of 100 and 250 ppm of initial PHE (as sole carbon and energy sources), incomplete PHE removals were observed at higher initial PHE concentrations up to 2000 ppm after 7 days. IC system resulted in the maximum PHE removal of 400 ppm at initial PHE concentration of 750 ppm and inoculum size of 600 mg l⁻¹, while under these conditions FC system removed 310 ppm of PHE. Moreover, bead cell density was shown to affect the performance of IC system, with the lower density of 5 mg ml⁻¹ leading to a higher PHE removal due to the enhanced transport phenomena in the culture. Additionally, a correlation was proposed to predict PHE biodegradation at a range of initial PHE concentrations.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P₆₈₀, and the first quinone electron acceptor, Q_A, were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S₂Q_A⁻ and S₂Q_B⁻ states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S₂Q_A⁻ and S₂Q_B⁻ states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q_A/Q_A⁻ relative to that observed in the presence of DCMU, charge recombination from the S₂Q_A⁻ state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P₆₈₀⁺Phe⁻ radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P₆₈₀^* from ¹[P₆₈₀⁺Ph⁻] and direct recombination of the ³[P₆₈₀⁺Ph⁻] and ¹[P₆₈₀⁺Ph⁻] radical states, respectively. An additional non-radiative pathway involves direct recombination of P₆₈₀⁺Q_A⁻. The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of ΔG(P₆₈₀^* ↔ P₆₈₀⁺Phe⁻) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of ΔG(P₆₈₀⁺Phe⁻ ↔ P₆₈₀⁺Q_A⁻) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Synthesis and characterization of a stable dispersion of multifunctional Gd2O3:Er,Yb in polyvinyl alcohol

A stable dispersion of multifunctional Gd₂O₃:Er,Yb phosphor in polyvinyl alcohol (PVA) was synthesized by varying the concentration of Yb³⁺ ions. It had a strong ultraviolet–visible‐near infrared (UV–vis) upconversion emission and applications in temperature and magnetic field sensors (e.g., nano‐heaters), as well as potential use in bioleveling and bioimaging. Stability of the dispersion was found to strongly depend on the mixing process of the powder in the polymer solution. Spherical shaped nanoparticles in cubic phase of ~ 43 nm diameter were synthesized and characterized by X‐ray diffraction; results were confirmed by scanning electron microscopy (SEM). Fourier transform infrared sspectroscopy (FT‐IR) and thermal analysis supported the presence of PVA. NIR pumping produced strong UC emission bands in the red and green regions extending up to very high UV (240 nm). This method provides an alternative for synthesizing a highly UC‐efficient non‐agglomerated pure transparent dispersion from various efficient phosphors for biological applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca²⁺, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.     

Delayed fluorescence from Rhodopseudomonas viridis following single flashes

Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studied using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P⁺) of the primary electron-donor (P) and the photoreduced form (X^?) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P⁺ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P⁺X^?, and (c) upon transfer of an electron from X^? to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P⁺ by cytochromes and the oxidation of X^? by Y, the decay kinetics of delayed fluorescence are identical with those of P⁺X^?, as measured from optical absorbance changes.The main decay route for P⁺X^? under these conditions has a rate-constant of approximately 10³ s^?1. In contrast, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P⁺X^? returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s^?1, at 295 °K and pH 7.8. The decay kinetics of P⁺X^? and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal · mol^?1. We conclude that the main decay route involves tunneling of an electron from X^? to P⁺, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P⁺X^? results in increases of both the energy and the entropy of the system, by 16.6 kcal · mol^?1 and 8.8 cal · mol^?1 · deg^?1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD⁺ and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD⁺ cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD⁺ binds in an extended conformation with a distance of 15 Å between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD⁺, with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD⁺-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.