Visualization of dioxygen bound to copper during enzyme catalysis.

X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal.

Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution.

BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ). RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms. CONCLUSIONS: The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Electrochemical screening of self-assembling beta-sheet peptides using supported phospholipid monolayers.

In the context of the medical applications of beta-sheet self-assembling peptides, it is important to be able to predict their activity at the biological membrane level. A study of the interaction of four systematically varied 11-residue (P11-1, P11-2, P11-6 and P11-7) and one 13-residue (P13-1) designed beta-sheet self-assembling peptides with DOPC monolayers on a mercury electrode is reported in this paper. Experiments were carried out in 0.1 mol dm(-3) KCl electrolyte with added phosphate buffer (0.001 mol dm(-3)) at pH approximately 7.6. The capacity-potential curves of the coated electrode in the presence and absence of the different peptides were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode surfaces in the presence and absence of the peptides was estimated between 65,000 and 0.1 Hz at -0.4V versus Ag/AgCl 3.5 mol(-3) dm(-3) KCl. The monolayer permeabilising properties of the peptides were studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. Of the five peptides studied, P11-2, P11-7 and P13-1 interact most strongly with the DOPC layer. P11-1 which has a polar primary structure shows no obvious interaction with the phospholipid but surprisingly, it permeabilises the phospholipid layer to Tl(+).

Cooperativity of the phase transition in single- and multibilayer lipid vesicles.

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites.

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies.

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.

Crystal structure of a free radical enzyme, galactose oxidase.

The crystal structure of the copper-containing enzyme, galactose oxidase, has been solved by multiple isomorphous replacement and refined to a resolution of 1.7 A. The X-ray structure reveals a unique polypeptide fold. The protein can be divided into three domains, all of which consist almost entirely of beta-strands. The structure of the second domain is particularly striking, 28 beta-strands arranged in a pseudo 7-fold symmetry. The copper site is on the surface of the protein and extremely rich in aromatic side-chains. The copper ion has two histidines, two tyrosines, and one external ligand in distorted square pyramidal coordination. The presence of pyrroloquinoline quinone as a covalently bound cofactor in GOase has been excluded. Instead, an unexpected covalent linkage between Tyr272 and Cys228 has been observed, whose functional role may relate to the presence of a tyrosine free radical at Tyr272. The tyrosine free radical could be stabilized by delocalization to Cys228 and stacking interactions with Trp290. A structural model for substrate binding is proposed that offers an explanation for the substrate specificity of the enzyme and many of the spectroscopic and enzymological data. Although the model lacks direct confirmation at present, it should provide a stimulus for further spectroscopic and crystallographic studies.

Status of the cofactor identity in copper oxidative enzymes.

Much conflicting data have appeared in the literature regarding the nature of the active site structures responsible for catalysis in three classes of copper enzymes: the copper amine oxidases, dopamine beta-monooxygenase and galactose oxidase. Although pyrroloquinoline quinone has been proposed to be the active site cofactor in each instance, new findings indicate this is not the case. Instead, recently available data indicate a spectrum of strategies for substrate activation, which range from direct metal catalysis (dopamine beta-monooxygenase) to the involvement of protein-derived radicals (galactose oxidase) and protein-derived quinones (copper amine oxidases).

Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein.

Equilibrium and non-equilibrium conformations of peptides in lipid bilayers.

A synthetic, hydrophobic, 27-amino-acid-residue peptide 'K27', modelled on the trans-membrane domain of the slow voltage-gated potassium channel, IsK, has been incorporated into a lipid bilayer and its conformational properties studied using FT-IR spectroscopy. The conformation following reconstitution is found to be dependent on the nature of the solvent employed. When the reconstitution is conducted by solvent evaporation from a methanol solution, aggregates comprised of beta-strands are stabilised and their concentration is essentially invariant with time. By contrast, when trifluoroethanol is used, the initial conformation of the peptide is alpha-helical. This then relaxes to an equilibrium state between alpha-helices and beta-strands. The alpha-helix-to beta-strand conversion rate is relatively slow, and this allows the kinetics to be studied by FT-IR spectroscopy. The reverse process is much slower but again can be demonstrated by FT-IR. Thus, it appears that a true equilibrium structure can only be achieved by starting with peptide in the alpha-helical conformation. We believe this result should be of general validity for hydrophobic peptide reconstitution. The implications for conformational changes in membrane proteins are discussed.

Protein-lipid interactions in cytochrome oxidase from Saccharomyces cerevisiae. Effects of detergents and reconstitution of enzyme activity by phospholipids by using cholate-mediated exchange.

Cytochrome oxidase, purified from the yeast Saccharomyces cerevisiae, was shown to have associated phospholipid, cholate or detergent, which could be varied by dialysis or (NH4)2SO4 precipitation of the protein. Cholate and the detergents Triton X-100 and Tween 80 were shown to differ in their ability to support enzyme activity. Changes in the Vmax, but not the Km, for ferrocytochrome c as the cholate concentration was varied indicate that cholate increases the number of exposed active sites of the enzyme. Cholate was used to introduce chosen phospholipids into the lipid environment of yeast cytochrome oxidase. Kinetic studies clearly showed that cholate can mediate exchange of exogenous for endogenous phospholipid. All phospholipids screened supported activity up to the basal value for the unsubstituted enzyme, whereas mitochondrial phosphatidylethanolamine and various phosphatidlycholines (except 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) produced enhanced activity. A detailed kinetic examination revealed that the major effect of phosphatidylethanolamine is to increase k+1, whereas the major effect of phosphatidylcholine is to increase K+2 in the minimal kinetic scheme E + S k+1 in equilibrium k-1 ES k+2 leads to E + P Cardiolipin, although supporting activity, does not give any enhancement of k+1 or k+2 over the values for the cholate control. The relevance of these observations to protein-lipid interactions in cytochrome oxidase is discussed.

A catalytic mechanism for benzylamine oxidase from pig plasma. Stopped-flow kinetic studies.

1. Ammonium ion is shown to decrease the rate constants for Schiff's base formation and formation of a reduced intermediate during the catalytic cycle of benzylamine oxidase from pig plasma. The rat constant for reoxidation of the reduced intermediate is also inhibited whilst the rate constant for conversion of the oxidised enzyme form back to native enzyme is stimulated by ammonium ion. 2. Ammonium ion changes the electron paramagnetic resonance spectrum of the cupric centres in the enzyme, indicating that ammonia binds to the copper. 3. A catalytic mechanism for benzylamine oxidase is proposed on the basis of these and other results. This mechanism includes a novel step in which a hydroxyl coordinated to copper acts as a nucleophyle to facilitate hydride ion transfer to oxygen during the reoxidation process.

Morphology of egg phosphatidylcholine-cholesterole single-bilayer vesicles, studied by freeze-etch electron microscopy.

Homogeneous, small, single-bilayer vesicles were prepared from egg phosphatidylcholine with various concentrations of cholesterol by ultrasonic dispersion in 0.1 M KCl, 0.01 M Tris, pH 8.0, buffer, followed by gel chromatography. The shape and size distributions of the fractionated vesicles were investigated for preparations with cholesterol compositions from 0 to 50 moles/100 moles, using freeze-etch electron microscopy. The size distribution was estimated from the shadow width of vesicles which were exposed by etching and the vesicle shape was checked by comparing the images obtained by tilting the replicas. The widths of the vesicle diameter distributions were relatively broad, corresponding to standard deviations in the range 60--90 A, but showing no systematic variation with cholesterol composition. In all cases it was found that 70% of the vesicle diameters lay within 150 A of the modal value. The apparent vesicle diameters remained constant for cholesterol compositions up to 20 moles/100 moles (modal diameter = 330 +/- 20 A, mean diameter = 350 +/- 3 A), but there was a sharp net increase in diameter at 30 moles cholesterol/100 moles reaching a model diameter of 430 +/- 20 A (mean diameter = 430 +/- 3 A) at 50 moles cholesterol/100 moles. Using the tilted microscope stage it was found that all vesicles were spherical at all cholesterol compositions studied, including those above 30 moles cholesterol/100 moles. The molecular mechanism by which cholesterol controls the vesicle size is discussed in terms of the asymmetric distribution of cholesterol across the vesicle bilayer.

Incorporation of cytochrome oxidase into cardiolipin bilayers and induction of nonlamellar phases.

Cytochrome oxidase from beef heart has been lipid-substituted with beef heart cardiolipin. The lipid phase behavior and protein aggregation state of the reconstituted complexes have been studied with 31P NMR, freeze-fracture electron microscopy, and saturation-transfer ESR of the spin-labeled protein. In the absence of salt, the lipid has a lamellar arrangement, and the protein is integrated and uniformly distributed in the membrane vesicles and undergoes rapid rotational diffusion. The presence of the protein stabilizes the cardiolipin lamellar phase against salt-induced transitions to the inverted hexagonal phase. The threshold salt concentration becomes higher and the extent of conversion becomes lower with decreasing lipid:protein ratio. In high salt, lamellar-phase lipid with integrated protein coexists with hexagonal-phase lipid free of protein, and the rotational diffusion of the protein is drastically reduced as a result of the high packing density.

Evaluation of spin resonance spectroscopy of red blood cell membranes to detect malignant hyperthermia susceptibility.

We have evaluated a spin labelled electron spin resonance technique to identify malignant hyperthermia susceptible (MHS) patients. We studied 19 patients, 10 MHS and nine MHN (normal), using the standard European procedure. We were unable to obtain any evidence that this technique could be used to diagnose MH susceptibility. Furthermore, there was no significant difference in the fluidity of the red blood cell membranes between the two groups, which would have been indicative of a generalized membrane abnormality in MH.

Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase.

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.

Rotational motion of yeast cytochrome oxidase in phosphatidylcholine complexes studied by saturation-transfer electron spin resonance.

Cytochrome oxidase from yeast has been covalently labeled with a nitroxide derivative of maleimide and reconstituted in lipid-substituted complexes with dimyristoyl-, dioleoyl-, or dielaidoyl-phosphatidylcholine. The rotational mobility of the enzyme in the complexes has been studied as a function of temperature and time, and of lipid/protein ratio, using saturation-transfer electron spin resonance spectroscopy. For complexes with dimyristoylphosphatidylcholine, the rotational mobility of the protein decreases abruptly below the gel-to-fluid-phase transition. This change is accompanied by a lateral segregation of the protein, as seen by freeze-fracture electron microscopy, and by an increase in the activation energy for the enzymatic activity. A time-dependent decrease in the rotational motion of the protein is observed on incubating at temperatures in the fluid phase of the lipid. This corresponds with a time-dependent loss of enzyme activity observed on incubation at temperatures in the fluid phase, but not at temperatures in the gel phase, over a period of 3 h. The rotational mobility decreases with increasing protein concentration in the complexes, both in the fluid and in the gel phases. The dependence of the protein mobility on lipid/protein ratio can be interpreted quantitatively in terms of the effect of increased random protein-protein contacts in the fluid phase. The maximum limiting rotational correlation time for the protein diffusion at high lipid/protein ratios in the fluid phase is tau R[[ approximately equal to 25 microseconds, suggesting that the protein is present as either a monomer or more probably a dimer in the reconstituted membrane.

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.