Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.     

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Vibrational circular dichroism of tetraphenylporphyrin in peptide complexes? A computational study

The Raman and absorption spectra of tetraphenylporphyrin (TPP) were calculated and compared to experiment. The computation was based on the harmonic molecular force field and electric tensors obtained ab initio at the BPW91/6-31G* level. Good agreement was found between experimental and calculated frequencies and intensities. In order to estimate whether induced optical activity in chiral complexes interferes with the signal of peptide vibrations, the vibrational circular dichroism (VCD) spectra of TPP were simulated. The magnetic field perturbation theory (MFP) and the gauge-invariant atomic orbitals (GIAO) were used for the simulation. Such spectra were compared to theoretical VCD intensities of a model tripeptide as well to experimental spectra of a complex of the peptide and tetrakis(p-sulfonatophenyl)porphyrin (TSPP). No significant contribution to VCD signal from the TPP residue was found in experimental spectra. Thus, possible peptide conformational changes occurring during the complexation can be monitored directly in the amide I frequency region.     

Intermolecular complexes between N-methyl-1,4-dihydronicotinamide and flavines. The influence of steric and electronic factors on complex formation and the rate of flavine-dependent dihydronicotinamide dehydrogenation.

The reaction of N-methyldihydronicotinamide (NMNH) with flavine analogs saturates at high dihydronicotinamide concentrations. Complex formation between the reactants depends mainly on steric but not on electronic factors. Thus flavine analogs that differ up to 243 mV in their oxidation-reduction potential vary only between 0.09 and 0.17 M in Kd. When the flavine plane becomes blocked by bulky substituents, however, complex stability decreases by more than an order of magnitude. NMNH-flavine complexes show long wave optical absorption. The energy of the long wave transition decreases with increasing oxidation-reduction potential of the flavine as expected for charge transfer complexes. The first-order rate constants of flavine-dependent dihydronicotinamide dehydrogenation increase with increasing oxidation-reduction potential of the flavine but they are almost independent of Kd. The reaction is not subject to general acid-base catalysis. Thus flavine-dependent dihydronicotinamide dehydrogenation may be interpreted to proceed via a charge transfer complex between oxidized flavine and reduced nicotinamide. In the rate-limiting conversion of the charge transfer complex into products hydrogen is transferred directly, the rate being governed by the difference in oxidation-reduction potential between flavine and dihydronicotinamide. An alternative mechanism where the observed charge transfer complex is not on the reaction pathway appears to be improbable but cannot be eliminated.     

Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase

Resonance Raman data are reported for the redox-activated form of galactose oxidase from Dactylium dendroides. Excitation within the red (659 nm) and blue (457.9 nm) absorption bands leads to strong resonance enhancement of ligated tyrosine vibrational modes at 550, 1170, 1247, 1484, and 1595 cm-1. The ring mode frequencies are unusually low, indicating a decreased bond order in the ring. The spectra clearly differ in both frequencies and relative intensities from those characteristic of known aromatic pi-radicals. Enhancement of tyrosine ring modes on excitation within absorption bands previously associated with the presence of the radical in the active site suggests that the ligated tyrosine residue is present in the radical site and may stabilize this radical species through formation of a charge transfer complex. A dramatically different Raman spectrum is observed for the N3- adduct of galactose oxidase, exhibiting a single strong 1483 cm-1 feature. The intense visible-near IR absorption bands for galactose oxidase may derive from transitions within a charge transfer complex between an aromatic free radical and a tyrosine-copper complex.     

Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase

Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.     

The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.     

Distinction between changes in membrane potential and surface charge upon chemotactic stimulation of Escherichia coli.

Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:6818-6825). The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane. The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis. To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response. All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude. This indicates the involvement of ion currents rather than alterations of local surface properties.     

Thiamine pyrophosphate biosynthesis and transport in the nematode Caenorhabditis elegans

Thiamine (vitamin B1) is required in the diet of animals, and thiamine deficiency leads to diseases such as beri-beri and the Wernicke-Korsakoff syndrome. Dietary thiamine (vitamin B1) consists mainly of thiamine pyrophosphate (TPP), which is transformed into thiamine by gastrointestinal phosphatases before absorption. It is believed that TPP itself cannot be transported across plasma membranes in significant amounts. We have identified a partial loss-of-function mutation in the Caenorhabditis elegans gene (tpk-1) that encodes thiamine pyrophosphokinase, which forms TPP from thiamine at the expense of ATP inside cells. The mutation slows physiological rhythms and the phenotype it produces can be rescued by TPP but not thiamine supplementation. tpk-1 functions cell nonautonomously, as the expression of wild-type tpk-1 in one tissue can rescue the function of other tissues that express only mutant tpk-1. These observations indicate that, in contrast to expectation from previous evidence, TPP can be transported across cell membranes. We also find that thiamine supplementation partially rescues the phenotype of partial loss-of-function mutants of the Na/K ATPase, providing genetic evidence that thiamine absorption, and/or redistribution from the absorbing cells, requires the full activity of this enzyme.     

On the orange color of Z-Trp-ONPo.

Out of all nitrophenyl esters of N(alpha)-protected alpha-amino acids Z-Trp-ONP(o) is the only one which is deep orange colored in the crystalline state. Any change in N(alpha)-protection, nature of amino acid, spatial separation between Trp and the ester-group or position of the nitro-substitutent in the aromatic ring of the ester function results in a loss of this characteristic property. We solved the molecular and crystal structure of Z-l-Trp-ONP(o) by X-ray diffraction analysis and investigated its color changes and visible (vis) and infrared (IR) absorption spectra in the solid state as a function of the amino acid derivative/KBr (w/w) ratio in the pellets. This investigation was extended to toluene solutions of different Z-Trp-ONP(o) concentrations by use of vis absorption and proton magnetic resonance spectroscopic techniques. The onset of the orange color correlates closely with the appearance of a concentration-dependent absorption band near 500 nm and concentration-dependent shifts of the urethane and indole NH proton resonances. Our observations can be explained by the formation of an intermolecular charge transfer complex involving the Trp indole and the -ONP(o) nitrophenyl as the donor and the acceptor moieties, respectively.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The insert within the catalytic domain of tripeptidyl-peptidase II is important for the formation of the active complex.

Tripeptidyl-peptidase II (TPP II) is a large (Mr>10(6)) tripeptide-releasing enzyme with an active site of the subtilisin-type. Compared with other subtilases, TPP II has a 200 amino-acid insertion between the catalytic Asp44 and His264 residues, and is active as an oligomeric complex. This study demonstrates that the insert is important for the formation of the active high-molecular mass complex. A recombinant human TPP II and a murine TPP II were found to display different complex-forming characteristics when over-expressed in human 293-cells; the human enzyme was mainly in a nonassociated, inactive state whereas the murine enzyme formed active oligomers. This was surprising because native human TPP II is purified from erythrocytes as an active oligomeric complex, and the amino-acid sequences of the human and murine enzymes were 96% identical. Using a combination of chimeras and a single point mutant, the amino acid responsible for this difference was identified as Arg252 in the recombinant human sequence, which corresponds to a glycine in the murine sequence. As Gly252 is conserved in all sequenced variants of TPP II, the recombinant enzyme with Arg252 is atypical. Nevertheless, as Arg252 evidently interferes with complex formation, and this residue is close to the catalytic His264, it may also explain why oligomerization influences enzyme activity. The exact mechanism for how the G252R substitution interferes with complex formation remains to be determined, but will be of importance for the understanding of the unique properties of TPP II.     

TREHALOSE SYNTHESIS IN EUGLENA GRACILIS (EUGLENOPHYCEAE) OCCURS THROUGH AN ENZYME COMPLEX1

The aim of this study was to isolate and characterize a trehalose‐synthesizing enzyme from Euglena gracilis Klebs. After purification by anion exchange chromatography, gel filtration, isoelectric focusing, and native electrophoresis, trehalose‐6‐phosphate synthase (TPS, EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (TPP, EC 3.1.3.12) activities could not be separated. Consequently, a TPS/TPP enzyme complex of about 250 kDa was suggested as responsible for trehalose synthesis in E. gracilis. The TPS activity was shown to be highly specific for glucose‐6‐P, and UDP‐Glc was the preferred glucose donor, but GDP‐Glc and CDP‐Glc could also act as TPS substrates. The TPP activity was highly specific for trehalose‐6‐P. In vitro phosphorylation assays revealed rapid decreases in TPS and TPP activities. These changes corresponded to variations in the elution profile of gel filtration chromatography after the phosphorylation treatment. Taken together, these results suggest that the proposed TPS/TPP complex might be regulated through a protein phosphorylation/dephosphorylation‐mediated mechanism that could affect the association state of the complex. Such a regulatory mechanism might lead to a rapid change in trehalose synthesis in response to variations in environmental conditions.     

Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin

For the purpose of the basic study of photodynamic therapy, the activity of the water-soluble P(V)porphyrin, dimethoxyP(V)tetraphenylporphyrin chloride (DMP(V)TPP), on photosensitized protein damage was examined. The quantum yield of singlet oxygen generation by DMP(V)TPP (0.64) was comparable with that of typical porphyrin photosensitizers. Absorption spectrum measurement demonstrated the binding interaction between DMP(V)TPP and human serum albumin, a water-soluble protein. Photo-irradiated DMP(V)TPP damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of DMP(V)TPP by human serum albumin supported the electron transfer mechanism. The estimated contribution of the electron transfer mechanism is 0.64. These results suggest that the activity of DMP(V)TPP can be preserved under lower oxygen concentration condition such as tumor.     

Effect of membrane mechanics on charge transfer by the membrane protein prestin

Prestin was found in the membrane of outer hair cells (OHCs) located in the cochlea of the mammalian inner ear. These cells convert changes in the membrane potential into dimensional changes and (if constrained) to an active electromechanical force. The OHCs provide the ear with the mechanism of amplification and frequency selectivity that is effective up to tens of kHz. Prestin is a crucial part of the motor complex driving OHCs. Other cells transfected with prestin acquire electromechanical properties similar to those in the native cell. While the mechanism of prestin has yet to be fully understood, the charge transfer is its critical component. Here we investigate the effect of the mechanics of the surrounding membrane on electric charge transfer by prestin. We simulate changes in the membrane mechanics via the corresponding changes in the free energy of the prestin system. The free energy gradient enters a Fokker-Planck equation that describes charge transfer in our model. We analyze the effects of changes in the membrane tension and membrane elastic moduli. In the case of OHC, we simulate changes in the longitudinal and/or circumferential stiffness of the cell’s orthotropic composite membrane. In the case of cells transfected with prestin, we vary the membrane areal modulus. As a result, we show the effects of the membrane mechanics on the probabilistic characteristics of prestin-associated charge transfer for both stationary and high-frequency conditions. We compare our computational results with the available experimental data and find good agreement with the experiment.     

Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c from Nostoc sp. PCC 7119

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions.     

Molecular orbital study on the interaction between coenzymes and enzymes; NAD+ or NADH and dehydrogenases

The absorption band at 260 mμ of NAD⁺ shifts to 360 mg by interaction with GAPDH or its analogues. Two explanations have been given on this red shift; one is an addition of such nucleophilic residue as sulfhydryl group in the enzyme to the position four in nicotinamide nucleus of NAD⁺, and the other is the charge transfer from such aromatic amino acid as tryptophan to NAD⁺. In the present paper, possibility of the charge transfer from indole residue to NAD⁺ was investigated quantum chemically. Taking into account of the electric field due to the charges in the enzyme, the absorption band of the NAD⁺-enzyme complex at 360 mμ was explained as a charge transfer from indole nucleus to NAD⁺. The blue shift of the absorption band of NADH at 340 mμ was also explained by taking into account of the electric field and this supported the proposition of Kosower (1962a).Stacking of adenine nucleus with indole nucleus in the NAD⁺-enzyme complex was suggested from the NMR spectroscopic data. Our molecular orbital calculations predicted that the effects of adenine on spectral shifts were not significant.     

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C₁₂TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C₁₀TEA and C₁₆TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C₁₂TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C₁₂TPP induced H⁺ permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663–668). C₁₂TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6 μM. Besides, C₁₂TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C₁₀TEA and C₁₆TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C₁₂TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.     

Induced absorption band of holotransketolase and its interpretation

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1"-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.