Investigation of dye/protein interaction and its application to enzyme purification

In this review the results of the interaction of the active dyes used in the USSR textile industry with microbial enzymes and blood serum proteins are discussed. The complexity of dye/protein interaction and the dependence of this interaction on different factors is demonstrated. Some practical aspects of the use of dye containing sorbents are presented and discussed. Their suitability for RNA ligase and DNA ligase, acetate kinase, alcohol dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase purification and blood serum protein fractionation is demonstrated.     

A membrane potential threshold for phage T4 DNA injection

DNA penetration from T4 phage adsorbed to $\text{Escherichia coli}$ was measured at different membrane potentials. There was a precipitous reduction in DNA penetration between 110 mV and 60 mV. This threshold of membrane potential for DNA penetration is independent of ΔpH and rather insensitive to external pH between 6 and 8.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

Reconstitution of biological molecular generators of electric current. H+-ATPase.

1. Generation of a transmembrane electric potential difference by oligomycin-sensitive ATPase complex, incorporated into spherical or planar phospholipid membrane, has been demonstrated. To this end, penetrating anion probe and direct voltmeter measurement of electric potential across phospholipid membrane were used. It was found that ATP-induced electric response is sensitive to oligomycin and protonophorous uncouplers. 2. The effect of variations in the phospholipid component of proteoliposomes on the electric generation was studied. It was revealed that the usage of mitochondrial phospholipids and phosphatidylethanolamine allows the highest values of membrane potential to be obtained in the case of ATPase proteoliposomes. In the case of cytochrome oxidase and bacteriorhodopsin proteoliposomes, phosphatidylserine was also shown to be quite suitable. Phosphatidylcholine was absolutely ineffective in all cases. 3. In proteoliposomes, containing both ATPase and bacteriorhodopsin, ATP and light induced generation of the electric field of the same direction. 4. In ATPase + cytochrome oxidase proteoliposomes, ATP hydrolysis and ascorbate oxidation was found to support electric generation of the same direction if cytochrome c was inside vesicles. Oxidation via external cytochrome c resulted in formation of electric field of the direction, opposite to that induced by ATP hydrolysis. 5. The data obtained in experiments with proteoliposomes of different types are discussed. The conclusion is made that conversion of energy of different resources into electric form is a common feature of membraneous energy transducers, which is in agreement with the Mitchellian principle of cellular energetics.     

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.     

Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans

Arginine 54 in subunit I of cytochrome c oxidase from Paracoccus denitrificans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a and lowers its midpoint redox potential [Kannt et al. (1999) J. Biol. Chem. 274, 37974-37981; Lee et al. (2000) Biochemistry 39, 2989-2996; Riistama et al. (2000) Biochim. Biophys. Acta 1456, 1-4]. During anaerobic reduction of the mutant enzyme, a small fraction of heme a is reduced first along with heme a(3), while most of heme a is reduced later. This suggests that electron transfer is impaired thermodynamically due to the low redox potential of heme a but that it still takes place from Cu(A) via heme a to the binuclear site as in wild-type enzyme, with no detectable bypass from Cu(A) directly to the binuclear site. Consistent with this, the proton translocation efficiency is unaffected at 1 H(+)/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully reduced enzyme reacts with O(2), the fast phase of membrane potential generation during the P(R )()--> F transition is unaffected by the mutation, whereas the slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-reduced mutant enzyme heme a remains oxidized due to its lowered midpoint potential, whereas Cu(A) and the binuclear site are reduced. In this case the reaction with O(2) proceeds via the P(M) state because transfer of the electron from Cu(A) to the binuclear site is delayed. The single phase of membrane potential generation in the 3e(-)-reduced mutant enzyme, which thus corresponds to the P(M)--> F transition, is decelerated, but its amplitude is comparable to that of the P(R)--> F transition. From this we conclude that the completely (4e(-)) reduced enzyme is fully capable of proton translocation.     

Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen

The reaction of cytochrome c oxidase with dioxygen has been studied by means of time-resolved measurements of electrical membrane potential (DeltaPsi). Microsecond time resolution was achieved by starting with the CO-inhibited enzyme, which was photolyzed after addition of oxygen. The time course of the reaction could be fitted by using a five-step sequential reaction as a model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacement across the membrane. After this lag, DeltaPsi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermediate, in a single phase, and conversion of F to O (the fully oxidized enzyme), in two phases. The amplitude of DeltaPsi was approximately equal for the P --> F and F --> O portions of the reaction. When the oxygen reaction is started with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, only the fast phase of DeltaPsi formation was observed, whereas no DeltaPsi was generated if the reaction was halted at P. This finding places the assignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectroscopic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the absence of oxygen ("backflow")-was studied. Parallel spectroscopic and electrometric measurements showed that the fast electron transfer from the low-spin heme to CuA in the backflow process results in approximately 11 times smaller amplitude of DeltaPsi as compared with DeltaPsi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to CuA amounts to movement of a unit charge across half of the membrane dielectric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxidative part of the reaction.     

Selecting neural networks for a committee decision

To improve recognition results, decisions of multiple neural networks can be aggregated into a committee decision. In contrast to the ordinary approach of utilizing all neural networks available to make a committee decision, we propose creating adaptive committees, which are specific for each input data point. A prediction network is used to identify classification neural networks to be fused for making a committee decision about a given input data point. The jth output value of the prediction network expresses the expectation level that the jth classification neural network will make a correct decision about the class label of a given input data point. The proposed technique is tested in three aggregation schemes, namely majority vote, averaging, and aggregation by the median rule and compared with the ordinary neural networks fusion approach. The effectiveness of the approach is demonstrated on two artificial and three real data sets.     

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.     

Electrogenic reactions of cytochrome bd

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.