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The critical review describes the known dicopper systems mediating the aromatic hydroxylation of monophenolic substrates. Such systems are of interest as structural and functional models of the type 3 copper enzyme tyrosinase, which catalyzes the ortho-hydroxylation of tyrosine to DOPA and the subsequent two-electron oxidation to dopaquinone. Small-molecule systems involving μ-η²:η² peroxo, bis-μ-oxo and trans-μ-1,2 peroxo dicopper cores are considered separately. These tyrosinase models are contrasted to copper–dioxygen systems inducing radical reactions, and the different mechanistic pathways are discussed. In addition to considering the stoichiometric conversion of phenolic substrates, the available catalytic systems are described. The second part of the review deals with tyrosinase. After an introduction on the occurrence and function of tyrosinases, several aspects of the chemical reactivity of this class of enzymes are described. The analogies between the small-molecule and the enzymatic system are considered, and the implications for the reaction pathway of tyrosinase are discussed (140 references).

This article summarizes the contributions given at the symposium "The Benefits of Gas-binding Proteins. Integrative and Evolutionary Physiology of Copper Proteins: Molecules to Organisms and their Environment," presented at the First International Congress of Respiratory Biology, August 14-16, at Bad Honnef/Bonn, Germany.

In some animals, the oxygen transport capacity of blood decreases when pH is lowered, yielding oxygen binding curves with Hill-coefficients smaller than unity. This so-called Root effect is observed in several fishes and is important for creating large oxygen partial pressures locally, for example in the swim bladder. While there is general agreement on the physiological advantages of this effect, its molecular basis remains ambiguous. Various studies show that isoforms of hemoglobins usually are present in the hemolymph, when the Root effect is observed. Here, we show that in such a case the mixture of these isoforms can exhibit apparent negative cooperativity, although each component taken separately can be described by the MWC model. In other cases, isolated isoforms exhibit true negative cooperativity. The well established MWC model describes many cooperative phenomena of enzymes and respiratory proteins but is not capable of describing negative cooperativity. In order to model negative cooperativity within a single molecular species a decoupling model might be employed, as pointed out previously. However, simulations show that it is not mandatory to have species with negative cooperativity, in order to obtain the binding curves typically seen for whole blood. These two aspects of the Root effect will be discussed on the basis of data from the literature.

The critical review describes the known dicopper systems mediating the aromatic hydroxylation of monophenolic substrates. Such systems are of interest as structural and functional models of the type 3 copper enzyme tyrosinase, which catalyzes the ortho-hydroxylation of tyrosine to DOPA and the subsequent two-electron oxidation to dopaquinone. Small-molecule systems involving μ-η²:η² peroxo, bis-μ-oxo and trans-μ-1,2 peroxo dicopper cores are considered separately. These tyrosinase models are contrasted to copper–dioxygen systems inducing radical reactions, and the different mechanistic pathways are discussed. In addition to considering the stoichiometric conversion of phenolic substrates, the available catalytic systems are described. The second part of the review deals with tyrosinase. After an introduction on the occurrence and function of tyrosinases, several aspects of the chemical reactivity of this class of enzymes are described. The analogies between the small-molecule and the enzymatic system are considered, and the implications for the reaction pathway of tyrosinase are discussed (140 references).

Oxygen, its nature and chemistry.- What is so special about this element? - A brief history of oxygen.- Coping with oxygen.- Aerobic metabolism - benefits from an oxygenated world.- Facilitated oxygen transport.- Climate over the ages is the environment stable? - Global warming: human intervention in world climate. - Oxygen in medicine.- Oxygen and the exploration of the universe.

In this paper, we report a study on the interaction of Eurypelma hemocyanin (Hc) with two fluorescent sulphydryl-reactive probes: N -(iodoacetylaminoethyl)-5-naphthylamino-1-sulfonic acid (AEDANS) and 5-dimethylaminona-phthalene-1-sulfonyl aziridin (DNS) which combine the high specificity of reactivity with the spectral properties of the naphthalenesulfonic acids. Both dyes form 1:1 stable complexes with the heterodimer bc , the other Eurypelma subunits being uninvolved in the binding. The labeled sulphydryl group is identified by means of the fluorescence properties of the labeled protein. Both dyes appear to accommodate in the same binding site which is located in the interior of the protein matrix, in close proximity to the copper active centers. An energy transfer process from Trp residues to the bound dyes is observed but only in copper-depleted protein: on this basis, a mean distance of ∼ 15–17 A between AEDANS or DNS groups and Trp-195 and Trp-197 (which are mostly responsible for the emission in apo-derivatives) is calculated. Based on the fluorescence properties of labeled protein, the only candidate which can bind the dye appears to be Cys-228. Based on the particular location of the protein-binding sites for AEDANS and DNS (only a distance of ∼ 10 A from the metal centers) and their peculiar fluorescence properties, both markers appear particularly suitable to monitor conformational transitions of arthropod Hc-active sites during oxygenation.

The tryptophan (Trp) fluorescence properties of the 24-meric hemocyanin (Hc) from the tarantula Eurypelma californicum and its subunits a, d and e were studied and compared with respect to the influence of the two copper atoms at the oxygen-binding site and the status of oxygenation. Fluorescence quenching experiments (by using acrylamide and iodide as external quenchers) and lifetime determinations suggested the occurrence of different classes of Trp responsible for the emission in oxygenated, deoxygenated and copper-depleted protein. On the basis of the different content and distribution of Trp in the isolated subunits and their position in the primary structure, each class of fluorophores was assigned to defined Trp residues. In 24-meric Hc, Trp-292 is mostly responsible for the fluorescence emission in the protein-oxygenated state. After oxygen removal, the major contribution probably arises from Trp-346 while in copper-depleted Hc, about ∼ 70% of fluorescence is due to Trp-195 and Trp-197. All these Trp are conserved in the different subunit types and appear to be located in the interior of the protein matrix, with the exception of Trp-292, which is partially exposed to the solvent. The ratio between the fluorescence quantum yields of oxy-, deoxy- and apo-24-meric Hc is ∼ 1:10:15. The drastic fluorescence decrease in oxy-Hc is mostly due to static quenching by copper ions on that classes of fluorophors (Trp-195, 197 and 346) localized very close to the active site (within 11 A). Each isolated subunit exhibits a peculiar fluorescence behavior, due to the heterogeneity of Trp distribution. In particular, the fluorescence properties of subunit d are dominated by Trp-386 which is present only in this subunit and is fully exposed on the protein surface.

As described in Chaps. 3 and 4, the advent of oxygenic photosynthesis triggered worldwide environmental changes. A world that had been reductive passed over into a state in which free dioxygen was available in the oceans and the atmosphere. We have already described the likely catastrophic effects on an anaerobic biota, but the changes were much broader than that. Dioxygen in the seas led to major changes in seawater chemistry. Iron, which had previously been soluble as ferrous salts, was precipitated in the ferric form. Copper, which had been insoluble in the anaerobic ocean as cuprous sulphide (Cu+-state), now became moderately soluble in the cupric form (Cu++-state).

Humankind has begun, in a tentative way, the immense project of exploring, and perhaps colonizing, other worlds. The grand enterprise has hardly begun and will certainly suffer many defeats and reversals, but it seems destined to go forward. In the course of this, both in seeking life in extraterrestrial environments and voyaging into them, we shall encounter a number of problems concerning the existence or provision of oxygen. The basis for this has been described in previous chapters. First, we would like to summarize arguments as to why life could have evolved on other planets. We need to know what to expect.

In the preceding chapter, we have emphasized the dangers that the advent of dioxygen presented to the existing anaerobic organisms, and the ways they evolved to deal with the problems. However, this is only part of the story and were it to have ended here, we and the world we know would not exist. What happened instead was quite remarkable; for life seized upon an opportunity presented by the presence of free dioxygen to become many-fold more efficient in extracting energy from foodstuffs. As we shall see, this aerobic, oxidative metabolism opened in turn a multitude of new opportunities for growth and diversification.

The implications of oxygen for medicine are basically of two kinds. First, there is the continued need by human tissues for an adequate supply of dioxygen; if that is not met, a condition called hypoxia may arise, with serious medical consequences. There are a wide number of causes for hypoxia, and a variety of medical responses.

It would seem that an introduction to oxygen is unnecessary, for we deal with it and depend upon it every moment of our lives. Oxygen is to us the essential stuff of the air we breathe. We are aerobic animals who obtain energy by oxidizing foodstuffs. As such, we are wholly dependent on oxygen for life – go without it for a couple of minutes and we panic and may even suffer irreversible brain damage. In a few more minutes, we perish. Animal metabolism depends upon oxygen for almost all of its energy-generating processes. Yet this was not always so. Early in the history of the Earth, there was essentially no free oxygen anywhere, although oxygen has always been one of the most abundant elements on Earth. In the early Earth, virtually all oxygen was bound in compounds, mainly water and silicate rocks. Primitive microbes managed life without free oxygen.

The amount of dioxygen an organism needs for aerobic metabolism depends on many factors, size and activity being the most important. However, as an approximate figure, we may say that a typical higher eukaryote will utilize about 3.5 ml dioxygen kg−1 body weight per minute. This must reach the tissues where active metabolism is occurring and be maintained there at a steady-state pressure of approximately 2 Torr. This will assure a sufficient rate of delivery to mitochondria and allow continued utilization therein for oxidative reactions (see Chap. 4). The problem faced by the organism is how to assure sufficient delivery to all the tissues, even those buried deep in the body, sometimes while inhabiting an oxygen-poor environment. There are a number of possible strategies to solve this problem. All have been used at one time or another in the course of evolution.

Sometime before 2.7 BYA, a new and biologically toxic substance began to appear in the environment. Biologically produced dioxygen, O2, probably first began to accumulate in small pools or layers above cyanobacterial mats. These photosynthesizers must have already developed ways to at least partially deal with dioxygen and, with greater difficulty, the reactive oxygen species (ROS) derived from it (see Chap. 1 and below). But for primitive anaerobes in the vicinity, these new substances must have been especially toxic. Nevertheless, it is clear that they evolved ways to cope with the new threats. One way was to simply avoid dioxygen altogether.

In the preceding chapter, we described climate changes that have occurred over very long geological periods. We concluded that Earth is currently in an interglacial interval within a rather long period of glaciations. Indeed, average carbon dioxide concentrations in the atmosphere have been slowly decreasing over the past 600,000 years, with accompanying cooling (Fig. 6.3). There have been, of course, many periodic changes in the CO2 concentrations and average temperature over this period (see Fig. 7.1). However, very recently, something quite unique and startling has occurred. As Fig. 7.1 shows, there has been a remarkable increase in CO2 levels, actually during the past 200 years, from 288 to 385 ppm today. Mean world temperature has shown an accompanying rise. This period is but an instant on any geological time scale, and it is very difficult to imagine any natural process that could account for it.

Where did oxygen come from? Remarkably, that atom of oxygen you have just breathed had its origin in the heart of an ancient star. To understand this, one has to make an imaginary journey back to the creation of the universe, the “big bang,” more than 12 BYA. We shall avoid details of physics, and simply describe a reasonable scenario that is accepted by most physicists today.

We present a modular, collaborative, open-source architecture for rigid body modelling based upon small angle scattering data, named sas_rigid. It is designed to provide a fast and extensible scripting interface using the easy-to-learn Python programming language. Features include rigid body modelling to result in static structures and three-dimensional probability densities using two different algorithms.

Haemocyanins are multimeric oxygen transport proteins, which bind oxygen to type 3 copper sites. Arthropod haemocyanins contain 75-kDa subunits, whereas molluscan haemocyanins contain 350–400-kDa subunits comprising seven or eight different 50 kDa FUs (functional units) designated FU-a to FU-h, each with an active site. FU-h possesses a tail of 100 amino acids not present in the other FUs. In the present study we show by X-ray crystallography that in FU-h of KLH1 (keyhole-limpet-haemocyanin isoform 1) the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain 3 in arthropod haemocyanin subunits has also recently been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both haemocyanin types once served to upload copper to the active site of the oxygen-binding domain.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.

Tyrosinases catalyze hydroxylation of monophenols to o-diphenols and their subsequent oxidation to o-quinones, whereas catecholoxidases catalyze only the latter reaction. Both enzymes occur in all organisms and are Type 3 copper proteins that perform the first steps of melanin formation. In arthropods, they play an essential role in the sclerotization of the exoskeleton. Very few phenoloxidases are characterized structurally or kinetically and the existence of an actual tyrosinase activity has not been demonstrated in most cases. Here we present for the first time a complete kinetic characterization of a tyrosinase from a crustacean (Palinurus elephas) including the influence of inhibitors. In contrast to most tyrosinases which are monomeric or dimeric, this tyrosinase occurs as a hexamer. However, the data did not indicate cooperativity in steady-state kinetics for the two substrates used, the monophenol tyramine and the diphenol dopamine. Mimosine as well as phenylthiourea (PTU) inhibited both monophenolhydroxylase and diphenoloxidase activity. Inhibition by mimosine was competitive, whereas PTU was a noncompetitive inhibitor. Furthermore, for the diphenolase activity substrate inhibition was observed, which was apparently abolished by adding PTU. These observations lead to the hypothesis that a secondary, allosteric binding site exists, which binds dopamine and PTU and reduces the catalytic activity.

Oxygen binding of hemocyanins results in an absorption band around 340nm and a strong quenching of the intrinsic tryptophan fluorescence. Our study analyses in detail the fluorescence quenching within two hemocyanins, a hexamer (Panulirus interruptus) and a 4 x 6-mer (Eurypelma californicum). Based on the comparison of calculated and measured transfer efficiencies we could show that: (1) For both hemocyanins FRET (fluorescence resonance energy transfer) is exclusively responsible for quenching of the tryptophan fluorescence upon oxygen binding. (2) Tryptophan quenching by FRET is independent of the oxy- or deoxy conformation of the protein. (3) The quenching takes place at the subunit level only and the oligomerization of both hemocyanins has no influence on the amount of quenching. Therefore, tryptophan fluorescence is a linear sensor for bound oxygen. It can be used as a model-free signal to investigate oxygen binding of hemocyanins at all aggregation levels. Furthermore it may provide a new way to analyse oxygen binding of phenoloxidases.

Homotropic and heterotropic allosteric interactions are important mechanisms that regulate protein function. These mechanisms depend on the ability of oligomeric protein complexes to adopt different conformations and to transmit conformation-linked signals from one subunit of the complex to the neighboring ones. An important step in understanding the regulation of protein function is to identify and characterize the conformations available to the protein complex. This task becomes increasingly challenging with increasing numbers of interacting binding sites. However, a large number of interacting binding sites allows for high homotropic interactions (cooperativity) and thus represents the most interesting case. Examples of very large, cooperative protein complexes are the giant hexagonal bilayer hemoglobins of annelid worms that contain 144 oxygen-binding sites. Moreover, these proteins show strict hierarchy in structure. In order to understand the interaction of various ligands such as oxygen, CO, or nitric oxide (NO), the principle binding behavior of these protein complexes has to be understood. For the hemoglobins of two species, the hierarchical structure is shown to have functional implications. By employing simultaneous analysis of several oxygen-binding curves, it could be shown that the nested MWC model provides a good description of the functional data. A strategy for the experimental setup and data analysis is suggested that allows for a reduction in the number of free parameters. Possible advantages of a hierarchical cooperative model compared to a linear extension of the MWC model are discussed.

Homotropic and heterotropic allosteric interactions are important mechanisms that regulate protein function. These mechanisms depend on the ability of oligomeric protein complexes to adopt different conformations and to transmit conformation-linked signals from one subunit of the complex to the neighboring ones. An important step in understanding the regulation of protein function is to identify and characterize the conformations available to the protein complex. This task becomes increasingly challenging with increasing numbers of interacting binding sites. However, a large number of interacting binding sites allows for high homotropic interactions (cooperativity) and thus represents the most interesting case. Examples of very large, cooperative protein complexes are the giant hexagonal bilayer hemoglobins of annelid worms that contain 144 oxygen-binding sites. Moreover, these proteins show strict hierarchy in structure. In order to understand the interaction of various ligands such as oxygen, CO, or nitric oxide (NO), the principle binding behavior of these protein complexes has to be understood. For the hemoglobins of two species, the hierarchical structure is shown to have functional implications. By employing simultaneous analysis of several oxygen-binding curves, it could be shown that the nested MWC model provides a good description of the functional data. A strategy for the experimental setup and data analysis is suggested that allows for a reduction in the number of free parameters. Possible advantages of a hierarchical cooperative model compared to a linear extension of the MWC model are discussed.

The structures of respiratory proteins such as hemoglobins and hemocyanins show obvious hierarchies. They belong to the class of allosteric macromolecules with cooperative functional properties, as do key metabolic enzymes. When examining the molecular mechanisms on which allostery and cooperativity are based, it could be shown for arthropod hemocyanins that the structural hierarchy of these macromolecules is reflected in a functional hierarchy. This relationship is described quantitatively in the "nesting" model. This model also offers explanations for the physiological significance of the prominent hierarchy of these molecules.

Continuous oxygen binding curves for two arthropodan hemocyanins were performed at different pH values ranging from 7.0 to 8.7 and in the presence of physiological concentrations of the bivalent ions Ca2+ and Mg2+. The arthropods Eurypelma californicum and Homarus americanus are classified as chelicerata and crustaceans, respectively. Their structurally well-characterized hemocyanins are composed of, in the case of E. californicum 24 subunits, and in the case of H. americanus 12 subunits. The role of protons as allosteric effectors of the oxygen binding was analysed in terms of the nesting model, which assumes hierarchies of allosteric equilibria that are based on obvious structural hierarchies. For each hemocyanin, the smallest structural repeating unit, the 12-mer or the 6-mer, respectively, was regarded as the "allosteric unit". Two allosteric units are allosterically coupled within the native molecules. The analysis revealed that in accordance with the postulations of the classical Monod-Wyman-Changeux model protons as allosteric effectors do not change the oxygen affinities of the four postulated conformations, but influence the allosteric equilibria between them at two different hierarchical levels. Model-independent determination of the affinity constants for the binding of the first and the last oxygen molecule to the native hemocyanins and to the isolated half-molecules confirmed the affinities calculated according to the nesting model. The stepwise establishment of new conformations during the assembly process from monomers to the structurally identical repeating unit and further on to the native molecule is shown. Possible physiological advantages of allosterically coupled allosteric units in contrast to allosterically uncoupled ones are thought to be (1) the option to regulate oxygen binding on different levels of structural hierarchy and (2) the increase of the oxygen-carrying capacity.

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.

High susceptibility of rabbit erythrocytes toward the pore-forming action of staphylococcal alpha-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for alpha-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules.

Copper proteins with dinuclear active sites comprise proteins with different structures and functions. The phenoloxidase, tyrosinase, and catecholoxidase are responsible for browning by starting the synthesis of melanin. These enzymes are involved in the primary immune response in invertebrates, plants, fungi as well as in the sclerotization of arthropods. The respiratory proteins hemocyanins are responsible for oxygen transport in some arthropods and molluscs. However, they can be converted to enzymes exhibiting phenoloxidase activity. Based on X-ray structures of hemocyanins and a catecholoxidase, large parts of folding motifs are very similar although the sequence identities are far below 30%. A model for the activation process of phenoloxidase activity is presented. Keywords: hemocyanin; tyrosinase; catecholoxidase; phenoloxidase; mollusc; arthropod

This paper describes a study performed to evaluate the feasibility of using a 1.5-T whole-body magnetic resonance imaging (MRI) equipment, in combination with pharmacokinetic modeling, to obtain in vivo information about the morphology and perfusion of tarantulas (Eurypelma californicum). MRI was performed on three tarantulas using spin-echo sequences for morphological imaging and a rapid spoiled gradient-echo sequence for dynamic imaging during and after contrast medium (CM; Gd-DTPA) injection. Signal enhancement in dynamic measurements was evaluated with a pharmacokinetic two-compartment model. Spin-echo images showed morphological structures well. Dynamic images were of sufficient quality and allowed a model analysis of CM kinetics, which provides information about regional perfusion. In conclusion, morphological and dynamic contrast-enhanced MRI of tarantulas is feasible with a conventional clinical scanner. Studies of this kind are therefore possible without a dedicated high-field animal scanner.

Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are important factors in wound healing and primary immune response. Their active site is characterized by a pair of antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a "type 3 copper centre" is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase). The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is still not completely understood, because nothing is known about their tertiary structure. One main reason for this deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-modified monomeric proteins with a low molecular mass of ca. 30kDa. They are secreted to the surrounding medium, where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is essential for the correct expression of the enzyme. This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get more insights in structure, enzymatic reactions and functions of "type 3 copper" proteins in general.

Publisher Summary The precise delivery of oxygen from respiratory surfaces to the tissues is mediated by cooperative and allosterically regulated carrier proteins, such as hemoglobin or hemocyanin. To establish cooperativity, these proteins must be able to adopt different conformations. These conformations are characterized by different ligand affinities, which have their basis in different structures as is the case for the deoxy and oxy states of human hemoglobin. To understand the cooperative interaction of these molecules at the molecular level, the structures of these conformations must be resolved and the transitions between them must be monitored. Because of the nature of sample preparation necessary for various methods, artificial forces may have an impact on the structure. X-ray structures are obtained from crystals. Electron microscopy delivers pictures from biomolecules either fixed on a surface and/or exposed to strong surface forces due to staining or ice buildup. These limitations also hold for newer techniques, such as atomic force microscopy (AFM). Two methods are available to study biomolecules in solution under in vivo conditions: high-resolution nuclear magnetic resonance (NMR) and small-angle scattering (SAS). NMR delivers structures with atomic resolution from rather small bio-molecules with a molecular mass less than 50 kDa. This is not the case for SAS. Here the resolution of the biomolecules obtained by this one- dimensional method is low but sufficient for large biomolecules. There are two methods of SAS— small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), each of which has its own advantages. SAXS experiments allow better angular resolution, whereas SANS enables the investigation of larger biomolecules. This chapter presents a methodical approach with SAS to obtain information about the large cooperative hemocyanins and their structural transitions on binding the ligand oxygen or allosteric effectors.

Hemocyanin sequences allineament shows the presence of highly invariant regions especially in the active site and in the tight intersubunits interaction sites. Comparing the aminoacids in contact regions between monomers is possible to interpret the stability of hexamers.

In the present study, we show the isolation and characterization of the protein haemoporin, which constitutes the second most abundant protein fraction in the haemolymph of the marine gastropod Aplysia californica. Although Aplysia is commonly used to investigate the molecular basis of learning, not much is known about the proteins in its haemolymph, which is in contact with the neurons owing to the open circulatory system of molluscs. In the native state, haemoporin is a macromolecular complex forming a cylinder with a central solvent-filled pore. The native complex most probably is a homopentamer made up from 70 kDa subunits with a molecular mass of 360 kDa and a sedimentation coefficient of 11.7 S. Prediction of the secondary structure by CD spectroscopy revealed that haemoporin contains 36% alpha-helices and 19% beta-strands. An absorption band in the 300-400 nm region indicates that haemoporin probably contains a bound substance. Haemoporin also contains a below average amount of tryptophan as evident from absorption and fluorescence spectra. The specific absorption coefficient at 280 nm (a (280 nm, 1 mg/ml)) varies between 0.42 and 0.59 l x g(-1) x cm(-1) depending on the method. The function of the protein is not yet known, but there are structural parallels between haemoporin and a pore protein reported previously in the haemolymph of another marine gastropod Megathura crenulata. The alanine-rich N-terminal sequence (AAVPEAAAEATAEAAPVSEF) is unique among protein sequences and indicates an alpha-helical structure. Whereas one side of the helix is hydrophobic and faces the interior of the protein, the other side contains a glutamic cluster, which may form the channel of the pore in the quaternary structure. Thus both proteins might belong to a new class of haemolymph proteins present in the haemolymph of marine gastropods.

There is a great need for research in the field of homeopathy for laboratory test systems to investigate the actions of ultrahighly diluted biological effectors. With this in mind, we used the luminescent bacterium Vibrio fischeri, which is used throughout the world in testing water quality. Luminescence inhibition is utilized as a test parameter for the toxicity of a sample. We used ultrahigh dilutions (UHD) of 3,5-dichlorophenol as effector and adapted the standard test procedure for water toxicity in a way that let us evaluate very minute effects. Three groups of samples were prepared and then blinded: 45 dilutions of 3,5-dichlorophenol in steps of 10, starting with 4.2×10−2 M, with vigorous shaking between dilution steps; 45 identical dilutions of 3,5-dichlorophenol without vigorous shaking; and 49 control samples of the diluent. The results of, and the discussion based on, a thorough statistical analysis led to the conclusion that an effect based on UHD, which results in an inhibition of luminescence of less than 1.5%, can be confirmed for some of the potency samples. There were both effective and ineffective samples in the three sample groups. The size of the effect was very small (ca. 1.5%), though statistically significant. The number of effective samples was significantly higher among the vigorously shaken samples than among the controls and the unshaken samples (14, 6 and 7 effective samples, respectively).

Hemocyanins are oxygen carriers of arthropods and molluscs. The oxygen is bound between two copper ions, forming a Cu(II)-O(2)(2-)-Cu(II) complex. The oxygenated active sites create two spectroscopic signals indicating the oxygen load of the hemocyanins: first, an absorption band at 340 nm which is due to a ligand-to-metal charge transfer complex, and second, a strong quenching of the intrinsic tryptophan fluorescence, the cause of which has not been definitively identified. We showed for the 4x6-mer hemocyanin of the tarantula Eurypelma californicum that the fluorescence quenching of oxygenated hemocyanin is caused exclusively by fluorescence resonance energy transfer (FRET). The tarantula hemocyanin consists of 24 subunits containing 148 tryptophans acting as donors and 24 active sites as acceptors. The donor-acceptor distances are determined on the basis of a closely related crystal structure of the horseshoe crab Limulus polyphemus hemocyanin subunit II (68-79% homology). Calculation of the expected fluorescence quenching and the measured transfer efficiency coincided extraordinary well, so that the fluorescence quenching of oxygenated tarantula hemocyanin can be completely explained by Forster transfer. This results explain for the first time, on a molecular basis, why fluorescence quantum yield can be used as an intrinsic signal for oxygen load of at least one arthropod hemocyanin, in particular that from the tarantula.

Hemoglobin from the leech Macrobdella decora belongs to the class of giant extracellular hexagonal bilayer globin structures found in annelid and vestimentiferan worms. These complexes consist of 144 heme-bearing subunits, exhibit a characteristic quaternary structure (2 × (6 × (3 × 4))), and contain tetramers as basic substructures that express cooperative oxygen binding and thus provide a structural basis for a hierarchy in allosteric interactions. A thorough analysis of the isolated tetramer indicates that it functions as a trimer of cooperatively interacting subunits and a non-cooperative monomer rather than as four interacting subunits. A thermodynamic analysis of the whole molecule favors the application of a nested Monod-Wyman-Changeux model with six cooperatively interacting 12-mer allosteric units. In contrast to the isolated tetramers, all subunits of the tetramers seem to be coupled cooperatively within the oligomerized 144-mer. Thus, besides hemocyanins and GroEL, the hexagonal bilayer hemoglobins represent another class of proteins in which the hierarchical quaternary structure provides the basis for nested interaction in their functional properties.

The effect of the chemical buffering component Tris (hydroxy-methyl-amino-methane) and of chloride ions on the oxygen binding of tarantula hemocyanin was studied at constant pH. It revealed that Tris at micromolar concentrations decreases the oxygen pressure at half-saturation (P50) by a factor of more than two, whereas chloride does not influence oxygen affinity. A thermodynamic analysis in terms of the nested model of allostery [(1987) Proc. Natl. Acad. Sci. 84, 1891-1895] indicated that Tris acts a an allosteric activator of oxygen binding by influencing the interaction between the 12-meric half-molecules of the 24-meric tarantula haemocyanin.

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

The respiratory protein of the keyhole limpet, Megathura crenulata, the hemocyanin (KLH), commonly used as an immunogen, binds oxygen cooperatively, which implies the existence of different conformations. For the first time, two different conformations of KLH1 were detected upon oxygenation, a deoxy and an oxy state, using small-angle neutron scattering. Rearrangements in the quaternary structure of KLH1 were predicted from the different radii of gyration and the shifts of the minima and maxima in the scattering curves. Upon oxygenation, KLH1 becomes smaller and more compact. Model reconstruction of KLH1 indicates a hollow cylinder with two rings located close to both ends, which move slightly together upon oxygenation.

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and l -lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2×6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2×6-meric hemocyanin of A. leptodactylus . The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (β and γ) are responsible for the two types of binding sites.