Mutation of alphaPhe55 of methylamine dehydrogenase alters the reorganization energy and electronic coupling for its electron transfer reaction with amicyanin

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of alphaPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the alphaF55A mutation also increases the rate of the true electron transfer (ET) reaction from O-quinol MADH to amicyanin. The reorganization energy associated with the ET reaction is decreased from 2.3 to 1.8 eV. The electronic coupling associated with the ET reaction is decreased from 12 to 3 cm(-1). The crystal structure of alphaF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-551i, has been determined. Little difference in the overall structure is seen, relative to the native complex; however, there are significant changes in the solvent content of the active site and substrate channel. The crystal structure of alphaF55A MADH has also been determined with phenylhydrazine covalently bound to TTQ in the active site. Phenylhydrazine binding significantly perturbs the orientation of the TTQ rings relative to each other. The ET results are discussed in the context of the new and old crystal structures of the native and mutant enzymes.     

Intramolecular electron transfer in [4Fe-4S] proteins: estimates of the reorganization energy and electronic coupling in Chromatium vinosum ferredoxin

The semi-classical electron transfer theory has been very successful in describing reactions occurring in biological systems, but the relevant parameters in the case of iron-sulfur proteins remain unknown. The recent discovery that 2[4Fe-4S] proteins homologous to Chromatium vinosum ferredoxin contain clusters with different reduction potentials now gives the opportunity to study the dependence of the intramolecular electron transfer rate between these clusters as a function of the driving force. This work shows how decreasing the reduction potential difference between the clusters by site-directed mutagenesis of C. vinosum ferredoxin modifies the rate of electron hopping between the two redox sites of the protein by measuring the line broadening of selected 1H NMR signals. Beside the shifts of the reduction potentials, no signs of large structural changes or of significant alterations of the intrinsic kinetic parameters among the different variants of C. vinosum ferredoxin have been found. A reorganization energy of less than 0.5 eV was deduced from the dependence of the electron transfer rates with the reduction potential difference. This small value is associated with a weak electronic coupling between the two closely spaced clusters. This set of parameters, determined for the first time in an iron-sulfur protein, may help to explain how efficient vectorial electron transfer occurs with a small driving force in the many enzymatic systems containing a 2[4Fe-4S] domain.     

Self-assembled complexes of oligopeptides and metalloporphyrins: measurements of the reorganization and electronic interaction energies for photoinduced electron-transfer reactions

Cationic porphyrins form ground state electrostatically associated complexes with anionic oligo-electrolytes such as those formed by a series of glutamic acid (E) residues. Temperature dependencies were measured of the rate constants for intra-complex electron transfer to the triplet state of Pd(II)TMPyP4+ from a tyrosine (tyr, Y) or tryptophan (trp, W) moiety connected to a glutamic acid tetramer. In complexes such as YE4, E2YE2, YE4G10E (G, glycine), and WE4 these data were used to estimate the reorganization energy (lambda) and electronic interaction energy (HDA) relevant to the process. For all tyr-peptide complexes, lambda values were found to be large (lambda approximately 1.60 +/- 0.06 eV), reflecting a relatively high medium polarity in the vicinity of tyr residues. It further indicates that the tyr residues in all oligo-peptides are exposed to the aqueous medium in a similar way irrespective of the position of the aromatic moiety in the peptide chain. A significantly lower lambda value (lambda = 1.08 eV) was derived for the tryptophan-containing peptide complex, indicating a relatively higher hydrophobic character of trp compared to tyr. The electronic coupling matrix elements (HDA) derived for tyr-peptide complexes (5.1 meV for YE4, 5.4 meV for YE4G10E and 7.5 meV for E2YE2) were larger than that found for WE4 (1.1 meV). Molecular dynamics calculations were employed to obtain structural features of the porphyrin-peptide complexes. These showed average distances between the center of mass (COM) of the porphyrin ring and the center of mass of the amino acid aromatic ring of 816 +/- 140 pm (YE4), 800 +/- 80 pm (E2YE2), 900 +/- 130 pm (YE4G10E) and 970 +/- 160 pm (WE4). The molecular dynamics calculations were shown to be in good agreement with the experimentally determined electronic interaction energies, strongly suggesting that HDA is primarily responsible for the dependence of the electron-transfer rate constant (KET) on the donor-acceptor separation distance and relative orientation. The higher HDA (7.55 meV) derived for tyr incorporated into the middle of the peptide backbone (E2YE2) was presumed to be associated with a higher degree of orbital overlap due to a more favorable ring-ring orientation. Overlap parameters (beta derived for all peptide-porphyrin complexes were similar (approximately 0.95 +/- 0.06 A-1), being in good agreement with most literature values for similar systems. Finally, the intra-complex electron-transfer ratio (ktrp/ktyr) derived from flash photolysis experiments and the corresponding ratio derived from Marcus' theory combined with experimental data from the temperature-dependence investigations and electrochemical measurements were found to be in excellent agreement. This same consistency was found for the couple E4Y and E2YE2. The empirical expression (Moser and Dutton) governing the intraprotein electron-transfer rate constant in native systems combined with our experimental data (kET, lambda, delta G0) yielded tunneling pathway distances in excellent agreement with those arising from the molecular modeling studies. The exception was for the long peptide YE4G10E, for which the Quenched Molecular Dynamic (QMD) sampling technique was complicated and is probably inadequate.     

Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix

The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.     

Voltammetric behavior of the MCF-7 cell cytoplasm and the effect of taxol on voltammetric response

The aims of this study were to find the electroactive species in the human breast cancer (MCF-7) cell cytoplasm causing a voltammetric response of the cells and to establish a simple and rapid measurement method to obtain strong and direct electrochemical responses objectively reflecting the cell viability. Ultrasonication was carried out for the electrochemical detection. The presence of guanine and xanthine in the MCF-7 cell eluent secreted by the living cells and in the MCF-7 cell cytoplasm was verified by HPLC assay with a DAD system and chemometric method. The concentrations of guanine and xanthine in the MCF-7 cell cytoplasm and the voltammetric response of the MCF-7 cell cytoplasm had higher levels than those of intact cell suspensions. Additionally, taxol caused a decrease of the voltammetric response of the cytoplasm and concentrations of xanthine and guanine in the cytoplasm. Therefore, the origin of the voltammetric response of the MCF-7 cytoplasm was driven by the alteration of the levels of xanthine and guanine, which were related to the cell viability. Thus, the voltammetric response of the ultrasonicated MCF-7 cell suspension could be used to monitor the MCF-7 cell growth and to evaluate the effectiveness of antitumor drugs on tumor suppression.     

Parallel mechanistic studies on the counterion effect of manganese salen and porphyrin complexes on olefin epoxidation by iodosylarenes

Counterions of manganese(III) porphyrin complexes influence diastereoselectivity in cis-stilbene epoxidation and product distribution in cyclohexene epoxidation markedly. In the epoxidation of cis-stilbene by iodosylbenzene carried out in a solvent mixture of CH(3)CN and CH(2)Cl(2), trans-stilbene oxide is the major product in the reaction of manganese complexes bearing a ligating anion (i.e., Cl(-)), whereas cis-stilbene oxide is the dominant product in the reactions of manganese complexes bearing a poorly-ligating anion (i.e., CF(3)SO(4)(-)). In cyclohexene epoxidation, the yields of allylic oxidation products such as cyclohexenol and cyclohexenone are higher when the counterion of the manganese catalysts is Cl(-) than when the counterion is CF(3)SO(4)(-). The product selectivities are also dependent on the nature of iodosylarenes and the axial and porphyrin ligands of the manganese porphyrin catalysts. The observation that product selectivities are different depending on the iodosylarenes may indicate the involvement of multiple oxidants in oxygen atom transfer reactions. These results are compared with those observed in manganese salen-catalyzed epoxidation of olefins by iodosylarenes.     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.     

Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.     

DNA-protein complexes of the nuclear matrix: visualization and partial characterization of the protein component

Complexes composed of DNA attached to the nuclear matrix and of proteins most tightly bound to DNA are visualized as globular particles 25-35 nm in diameter. Their morphology depends greatly on the isolation conditions: a Cs salts/urea combination permits the isolation while CsCl/sarcosyl destroys the particles. The preparation is shown to have the same protein content regardless of the treatment employed. The proteins of the complex are resistant to SDS and pronase treatment and to phenol/chloroform extraction while being associated with DNA.     

Syntheses of some new methylcyclopentadienylmolybdenum complexes: Characterizations, crystal structures and comparisons with related complexes

As part of a long-term study of the substitution reactions of piano-stool type cyclopentadienylmetal carbonyl complexes, several new methylcyclopentadienylmolybdenum compounds have been prepared and characterized by methods including IR spectroscopy, electrospray ionization mass spectrometry and X-ray crystallography. The complexes reported here include [{Cp′Mo(CO)₃}₂I]BPh₄, cis-Cp′Mo(CO)₂(PPh₃)I and [Cp′Mo(CO)₃(CH₃CN)]BF₄ (Cp′ = η⁵-C₅H₄CH₃). In addition to their syntheses, comparisons are made between their IR spectroscopic and X-ray crystal structure data and those of similar complexes.     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450

In order to examine the reactivity of active intermediate derived form iron porphyrins, competitive oxidations of alkane and alkene were carried out. It has been proposed that the first step of alkane hydroxylation is H atom abstraction and that of alkene is one-electron transfer. Therefore, it is expected that alkene-alkane competitive oxidation can be used as a probe for discrimination of differences in chemical properties among active species. Cytochrome P450 and SR complex, which is a stable thiolate-ligated iron porphyrin, mediated the oxidation of alkane much more preferentially than iron porphyrin coordinated by imidazole or chloride. These results indicate that thiolate coordination alters the reactivity of the two-electron-oxidized intermediate in a manner that is much more favorable to alkane hydroxylation than the case of chloride or imidazole coordination.     

Systematic development of computational models for the catalytic site in galactose oxidase: impact of outer-sphere residues on the geometric and electronic structures

A systematic in silico approach has been employed to generate sound, experimentally validated active-site models for galactose oxidase (GO) using a hybrid density functional, B(38HF)P86. GO displays three distinct oxidation states: oxidized [Cu(II)-Y*]; semireduced [Cu(II)-Y]; and reduced [Cu(I)-Y]. Only the [Cu(II)-Y*] and the [Cu(I)-Y] states are assumed to be involved in the catalytic cycle, but their structures have not yet been determined. We have developed several models (1-7) for the [Cu(II)-Y*] state that were evaluated by comparison of our computational results with experimental data. An extended model system (6) that includes solvent molecules and second coordination sphere residues (R330, Y405, and W290) is essential to obtain an experimentally correct electronic structure of the active site. The optimized structure of 6 resulted in a five-coordinate Cu site with a protein radical centered on the Tyr-Cys cofactor. We further validated our converged model with the largest model (7) that included additional outer-sphere residues (Q406, H334, Y329, G513, and T580) and water molecules. Adding these residues did not affect significantly the active site's electronic and geometric structures. Using both 6 and 7, we explored the redox dependence of the active-site structure. We obtained four- and three-coordinate Cu sites for [Cu(II)-Y] and [Cu(I)-Y] states, respectively, that corroborate well with the experimental data. The relative energies of these states were validated by a comparison with experimental redox potentials. Collectively, our computational GO models well reproduce the physicochemical characteristics of the individual states, including their redox behaviors.     

Synthesis and spectral luminescent characteristics of the porphyrin complexes with the platinum group metals

The syntheses of complexes of natural and synthetic porphyrins with Pt, Pd, Rh, and Ru are reported. Their electronic absorption spectra, phosphorescence spectra, and lifetimes at room temperature both in the presence and in the absence of oxygen were studied. Variations in the nature of the central metal atom and in the substituents in pyrrol and phenyl rings allow the obtaining of metalloporphyrins phosphorescing at room temperature with various phosphorescence excitation and emission spectra.     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Analysis on sliding helices and strands in protein structural comparisons: a case study with protein kinases

Protein structural alignments are generally considered as 'golden standard' for the alignment at the level of amino acid residues. In this study we have compared the quality of pairwise and multiple structural alignments of about 5900 homologous proteins from 718 families of known 3-D structures. We observe shifts in the alignment of regular secondary structural elements (helices and strands) between pairwise and multiple structural alignments. The differences between pairwise and multiple structural alignments within helical and beta-strand regions often correspond to 4 and 2 residue positions respectively. Such shifts correspond approximately to "one turn" of these regular secondary structures. We have performed manual analysis explicitly on the family of protein kinases. We note shifts of one or two turns in helix-helix alignments obtained using pairwise and multiple structural alignments. Investigations on the quality of the equivalent helix-helix, strand-strand pairs in terms of their residue side-chain accessibilities have been made. Our results indicate that the quality of the pairwise alignments is comparable to that of the multiple structural alignments and, in fact, is often better. We propose that pairwise alignment of protein structures should also be used in formulation of methods for structure prediction and evolutionary analysis.